Adrenocorticotropic hormone analogs and related methods

ABSTRACT

ACTH analog compounds of the present invention include compounds comprising an ACTH peptide sequence with one or more structural modifications that can have one or more of the following preferred ACTH analog biological functions: (1) reduction of corticosteroid secretion by adrenal membrane in the presence of the ACTH analog compared to unmodified ACTH, (2) reduction of corticosteroid secretion by adrenal membrane in the presence of endogenous ACTH and (3) increased MC-2R binding affinity with reduced activation of the MC-2R receptor compared to unmodified ACTH binding to the MC-2R melanocortin. The ACTH analog compounds of the present invention are therefore useful for treatment or prevention of diseases and disorders related to ACTH, ACTH receptors or corticosteroid secretion, such as premature labor and Cushing&#39;s Disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/622,436 entitled “Compositions and Methods forthe Treatment of Premature Labor, Cushing's Syndrome and RelatedDisorders,” filed Oct. 27, 2004, the entire contents of which areincorporated herein by reference.

GOVERNMENT SUPPORT

The subject matter of this application has been supported by a researchgrant from the National Institutes of Health (Grant Number NIH: DK50870)and a grant from the National Science Foundation (Grant Number NSFIBN-0132210). Accordingly, the government may have certain rights inthis invention.

TECHNICAL FIELD

The present invention relates to ACTH analog compounds, as well asrelated pharmaceutical compositions and methods of treatment.

BACKGROUND

Corticotropin, also known as adrenocorticotropic hormone (ACTH) is aprimary hormone secreted by the pituitary gland, that is believed to bea mediator in the production of a variety of vital growth andphysiological control steroids. ACTH stimulates the adrenal cortex. Morespecifically, it stimulates secretion of glucocorticoids such ascortisol in humans (or corticosterone in rodents), and has littlecontrol over secretion of aldosterone, the other major steroid hormonefrom the adrenal cortex. ACTH binds to the MC-2R adrenocorticotropichormone receptor expressed in the adrenal gland.

ACTH is secreted from the anterior pituitary in response tocorticotropin-releasing hormone (CRH) from the hypothalamus. Within thepituitary gland, ACTH is derived from a large precursor moleculepro-opiomelanocortin (POMC) that is cleaved by the action of specificpeptidase enzymes. The effects of ACTH on steroid synthesis can includean increase cholesterol esterase, the transport of cholesterol to andacross the mitochondrial membrane, cholesterol binding to P450SCC and,hence, an increase in pregnenolone production (see Nussey, S. and S.Whitehead, Endocrinology: An Integrated Approach, BIOS ScientificPublishers Ltd. (2001)). Subsequent actions can include the induction ofsteroidogenic enzymes and conspicuous structural changes characterizedby hypervascularization, cellular hypertrophy and hyperplasia. This isparticularly notable in conditions where excess ACTH can be undesirablysecreted over prolonged periods of time.

The steroid glucocorticoid is produced by adrenal fasciculata-reticulacells in the adrenal glands, and is secreted in response to an increasein the level of plasma adrenocorticotropic hormone (ACTH).Glucocorticoids are involved in carbohydrate, protein, and fatmetabolism, have been shown to have anti-inflammatory properties, andare hypersecreted during stress. In excess, glucocorticoids have beenshown to damage the hippocampus, a region of the limbic system of thebrain that is critical to cognitive functions such as learning andmemory. See, e.g., Sapolsky, R. M., Ann. N.Y. Acad. Sci. 746:294 (1994);and McEwen, B. S., Ann. N.Y. Acad. Sci. 746:134 (1994). Furthermore,glucocorticoid neurotoxicity and neuroendangerment has been shown to becritical in neural development and aging as well as in neurologicaldiseases related to hippocampal damage. See, e.g., deKloet, E. R., etal., Ann. N.Y. Acad. Sci. 746:8 (1994)

Corticosteroids are steroid hormones related structurally tocholesterol. These hormones are synthesized in the adrenal cortex andinclude the glucocorticoids (e.g. corticosteroids), themineralocorticoids (e.g aldosterone) as well as weak androgens andestrogens. The adrenal function, like that of the thyroid gland, isunder the control of the hypothalamus (HPT) and the pituitary (PIT).When corticosteroids (the naturally-occurring glucocorticoid) levelsdrop below a setpoint, the hypothalamus releases CRH (corticotropinreleasing hormone) which stimulates adrenocorticotropic hormone (ACTH)release from the pituitary. ACTH is a tropic hormone which stimulatesthe synthesis and secretion of corticosteroid (it has minimal effects onaldosterone synthesis/secretion), and the growth of the adrenal gland.

There is a need for compounds that bind to ACTH receptors with reducedactivation of corticosteroid secretion, for example to treatACTH-related conditions including: Cushing's Syndrome, impaired immuneresponse as a result of hypersecretion of corticosteroid, and certainadrenal-related causes of premature labor.

Cushing's syndrome is a disorder resulting from increased adrenocorticalsecretion of corticosteroid. Hyperfunction of the adrenal cortex may beACTH-dependent or it may be independent of ACTH regulation, e.g.production of corticosteroid by an adrenocortical adenoma or carcinoma.A common cause of Cushing's syndrome is excessive production of ACTH bythe pituitary gland. This elevated level of ACTH in the bloodstreamtypically is produced by a pituitary adenoma (Cushing's disease), but inrare instances has a different etiology. Cushing's syndrome resultingfrom the production of ACTH in a location other than the pituitary glandis known as ectopic Cushing's syndrome. Examples of ectopic sitesinclude thymoma, medullary carcinoma of the thyroid, pheochromocytoma,islet cell tumors of the pancreas and oat cell carcinoma of the lung.The overwhelming majority of Cushing's syndrome cases in humans,however, trace their etiology to a pituitary adenoma. Symptoms ofCushing's syndrome include weight gain, central obesity, steroidhypersecretion, elevated urinary cortisol excretion, moon face,weakness, fatigue, backache, headache, impotence, mental status changes,muscle atrophy, and increased thirst and urination compared to mammalsnot suffering from this disease. Diagnosis and treatment of Cushing'ssyndrome remains a challenge (see Oldfield, E. W. et al., N. Engl. J.Med., 325:897–905 (1991); Findling, J. W. et al., “Diagnosis anddifferential diagnosis of Cushing's syndrome,” Endocrinol. Metab. Clin.North Am., 30:729–47 (2001); Orth, D. N., “Cushing's syndrome,” N Engl JMed., 332:791–803 (1995)). No medical therapies are currently availablefor Cushing's syndrome. In experienced specialized centers, surgicalresection of ACTH-secreting pituitary microadenomas offers an overallcure rate of about 70–80%, but for macroadenomas cure rates onlyapproximate 30%, and the extensive surgical resection required portendssignificant risk to surrounding normal pituitary tissue, leading topartial or total hypopituitarism in about 80% of cases (Simmons, N. E.et al., “Serum Cortisol response to transphenoidal surgery for Cushingdisease,” J. Neurosurg., 95:1–8 (2001); Mampalam, T. J. et al.,“Transsphenoidal microsurgery for Cushing's disease: A report of 216cases,” Ann. Intern. Med., 109:487–93 (1988); and Trainer, P. J. et al.,“Transsphenoidal resection in Cushing's disease: undetectable serumcortisol as the definition of successful treatment,” Clin. Endocrinol.,38:73–8 (1993)). Thus, there is also a need for a treatment forCushing's syndrome where the source of ACTH is a disseminated pituitarytumor or an ectopic source that is effective and does not pose a risk tothe patient.

Compounds that bind to ACTH receptors with reduced activation ofcortisol secretion can also be used, for example, in treating thehypothalamus-pituitary-adrenal axis for initiation of pre-term labor.Preterm labor occurs in approximately 7–10% of all births andcontributes to a substantial proportion of perinatal morbidity andmortality (McCormick, M. C., “The contribution of low birth weight toinfant mortality and childhood morbidity,” N Engl J Med., 312:82–90(1985)). Preventing spontaneous abortion and premature labor, andprolonging gestation in human females, are desirable for many reasons.Gestation is desirably prolonged in order to (i) make more probable aviable live birth, (ii) reduce the incidence of health complicationsattending a prematurely born child, and (iii) reduce the time periodduring which a premature infant, even if healthy, must, because of itssize and viability, receive extraordinary care. All the factors of (i)live birth, (ii) a healthy child, and (iii) a child that can timelyleave the hospital in the custody of its parent(s), desirably impact thehappiness and well-being of the parents and relatives. There is also animpact on society from premature births, including a very great societaleconomic impact in caring for children who are delivered greatlyprematurely.

Agriculture and aquaculture can be more cost-effective when theorganisms can be raised at high population density. However, amongmammals, fowl and fish this frequently results in the over production ofadrenal stress hormones with deleterious consequences, includingimpaired immune function and decreased growth. A method for decreasingthe levels of adrenal stress hormones in these or other conditionscaused by prolonged stress and resulting in undesirable health changes,e.g. decreased immune function and susceptibility to disease, would alsobe desirable.

Various compositions and methods can be used to reduce ACTH levels, forexample through certain receptors for arginine vasopressin (AVP). U.S.Pat. No. 6,380,155, filed May 3, 2000, relates to the use of certainvasopressin receptor antagonist compositions for the regulation of ACTHrelease. Compositions, to treat ACTH-related conditions that regulateACTH levels are desirable, such as compositions that can bind to ACTHMC-2R receptors, while reducing or eliminating ACTH-inducedcorticosteroid production so as to mitigate undesirable conditionsassociated with elevated ACTH levels.

SUMMARY

Various modified adrenocorticotropic hormone (ACTH) peptides (“ACTHanalogs”) are provided that reduce or eliminate ACTH-inducedcorticosteroid secretion compared to unmodified ACTH. Preferably, theACTH analogs also reduce the secretion of corticosteroid from adrenalmembrane in the presence of unmodified ACTH.

The ACTH analog compounds preferably comprise at least amino acids 1–24of unmodified ACTH, with one or more amino acid substitutions. The ACTHanalog compounds can include one or more amino acid substitutions ortruncation with respect to the unmodified human ACTH amino acidsequence. Unmodified human ACTH is a polypeptide that has 39 amino acidresidues in the following sequence:N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰-Lys-Val-Tyr-Pro-Asn²⁵-Gly-Ala-Glu-Asp-Glu³⁰-Ser-Ala-Glu-Ala-Phe³⁵-Pro-Leu-Glu-Phe³⁹-Ac(SEQ ID NO. 1), where N and Ac represent the amino and carboxy terminalends, respectively, of the molecule. ACTH analogs can also comprisecompounds that include a peptide having one or more substitutions ormodifications of the sequence:N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰-Lys-Val-Tyr-Pro-Ac(SEQ ID NO: 2), where N and Ac represent the amino and carboxy terminalends, respectively, of the molecule. Preferred ACTH analog compoundsinclude at least amino acid residues 1–19 of hACTH or mACTH, morepreferably amino acid residues 1–24 of hACTH or mACTH, with one or moreamino acid substitutions.

ACTH analog compounds include compounds comprising the ACTH sequence ofSEQ ID NO:1 or SEQ ID NO:2 with one or more structural modificationsthat result in one or more of the following preferred ACTH analogbiological functions: (1) reduction of corticosteroid secretion byadrenal membrane in the presence of the ACTH analog compared tounmodified ACTH, (2) reduction of corticosteroid secretion by adrenalmembrane in the presence of endogenous ACTH and (3) increased MC-2Rbinding affinity with reduced activation of the MC-2R receptor comparedto unmodified ACTH. Examples of preferred ACTH amino acid substitutionsto form ACTH analog compounds include one or more of the following: (1)substitutions of one or more amino acid residues at positions 1–13 thatconserve the amino acids at positions 6–9 and/or promote or preserveMC-2R binding, (2) substitution of one or more amino acid residues atpositions 15–18 that prevent or antagonize enzymatic cleavage at thesepositions, (3) substitution of one or more amino acid residues atpositions 15–18 such that the ACTH analog does not comprise adjacentamino acid residues with basic side chains at positions 15–18, (4)substitution or truncation of one or more amino acid residues atpositions 20–24 that extend the serum half life of the ACTH analog, (5)substitution of one or more amino acid residues at positions 20–36 thatresult in an ACTH analog that reduces the corticosteroid secretion ofadrenal membrane in the presence of the ACTH analog compound compared toan unmodified ACTH peptide, (6) substitution or preferably truncation ofone or more amino acid residues at positions 25–39 that provide ACTHanalog compounds with desired release properties, or (7) truncation ofamino acid residues 25–39, with the amino acid residue at position 24forming the carboxy terminus of the molecule.

The ACTH analog compounds preferably bind to ACTH receptors such as themelanocortin 2 receptor (MC-2R) in adrenal membrane. More preferably,the ACTH analog compounds do not activate, or weakly activate, cellsexpressing MC-2R and inhibit or reduce the action of unmodifiedendogenous ACTH.

In a first embodiment, various ACTH analog compounds can include one ormore amino acid substitutions or truncation with respect to theunmodified human ACTH amino acid sequence. For example, a compositioncomprising an isolated ACTH analog peptide can include a peptide of SEQID NO:2 with at least one of the following amino acid substitutions:

a. the substitution of the Pro residue at position 19 of SEQ ID NO:2with the amino acid Trp; or

b. one or more amino acid substitutions of residues selected from aminoacid residues 16 to 18 of SEQ ID NO:2, such that the amino acid residues16, 17 and 18 of the ACTH analog do not include any two adjacent aminoacid residues selected from the group consisting of: Lys and Arg; andthe one or more amino acid residues substituted at position 16, 17 or 18of SEQ ID NO:2 are selected from the group consisting of: Lys, Arg, Gln,Gly, Ala, Val, Leu, Ile and an amino acid analog having an alkyl sidechain (such as Nle). Optionally, the ACTH analog peptide can include atleast one Ala, Gly, or another amino acid with an alkyl side chain(i.e., Val, Leu, Ile, an amino acid analog comprising an alkyl sidechain such as Nle), and at least one Arg residue substituted at any twoof the amino acid positions 15, 16, 17 or 18 of SEQ ID NO:2. The ACTHanalog can optionally consist essentially of the sequence of SEQ ID NO:2with the following amino acid substitutions: the Pro residue at position19 of SEQ ID NO:2 is substituted with the amino acid Trp; the amino acidat position 15 of SEQ ID NO:2 is selected from the group consisting of:Lys, Ala and Gln; and the ACTH analog peptide can comprise one or moreamino acid substitutions of residues selected from amino acid residues16 to 18 of SEQ ID NO:2, such that the amino acid residues 16, 17 and 18of the ACTH analog do not include any two adjacent amino acid residuesselected from the group consisting of: Lys and Arg. Preferably, the ACTHanalog peptide includes the amino acid sequence of SEQ ID NO:4 at aminoacid residues 6, 7, 8 and 9. The ACTH analog peptide can also includethe amino acid sequence of SEQ ID NO:12 at amino acid residues 15–19.

The first embodiment also includes ACTH analogs having the peptide ofSEQ ID NO:1 with at least one amino acid substitution, such assubstitution of an amino acid residue at position 19, 26, 30 or 36 ofSEQ ID NO:1. The ACTH analog peptide can also include the peptide of SEQID NO:1 with at least one of the following amino acid substitutions:

a. substitution of the Pro residue at position 19 of SEQ ID NO:1 withthe amino acid Trp or

b. one or more amino acid substitutions of residues selected from aminoacid residues 16 to 18 of SEQ ID NO:1, such that the amino acid residues16, 17 and 18 of the ACTH analog do not include any two adjacent aminoacid residues selected from the group consisting of: Lys and Arg; andthe one or more amino acid residues substituted at position 16, 17 or 18of SEQ ID NO:2 is selected from the group consisting of: Lys, Arg, Gln,Gly, Ala, Val, Leu, lie and Nle (or another amino acid analog having analkyl side chain).

Other preferred ACTH analog peptides include modifications of peptidesof SEQ ID NO:1 or SEQ ID NO:2 such that the ACTH analog peptide includesthe amino acid sequence of SEQ ID NO:6 at amino acid residues 15–19.ACTH analog peptides also include truncation of one or more peptidesACTH analog further comprises truncation of amino acid residues 25–39 ofSEQ ID NO:1. ACTH analog peptides of SEQ ID NO:20 are particularlypreferred.

In some embodiments, the administration of an ACTH analog peptidereduces the ACTH induced production of corticosterone by adrenalmembrane in an in vitro Serum Corticosteroid Induction Assay by at least10%, and preferably up to 100%, compared to the peptide of SEQ ID NO:2.For instance, in a second embodiment, ACTH analogs are modified ACTHpeptides that function to reduce corticosteroid secretion by adrenalmembrane in the presence of the ACTH analog compared to unmodified ACTH.

In some embodiments, the administration of the ACTH analog peptide in anin vivo Serum Corticosteroid Inhibition Assay reduces ACTH-inducedcorticosteroid secretion by at least a 10%. In a third embodiment, ACTHanalogs are modified ACTH peptides that function to reducecorticosteroid secretion by adrenal membrane in the presence ofendogenous ACTH.

In some embodiments, compositions are provided that include an isolatedACTH analog peptide of SEQ ID NO:2 with at least one amino acidsubstitution, wherein the ACTH analog peptide binds to and displaces apeptide of SEQ ID NO:2 from adrenal membrane. The peptide binding can bemeasured by an in vitro Serum-Free Adrenal Competitive Binding Assay.For example, in a fourth embodiment, ACTH analogs are modified ACTHpeptides that function to bind to adrenal ACTH receptors, such as theMC-2R receptor, preferably with increased MC-2R binding affinity andreduced activation of the MC-2R receptor compared to unmodified ACTH.Preferably, the ACTH analog peptide can bind to and displace a peptideof SEQ ID NO:2 from adrenal membrane, where the peptide binding ismeasured by an in vitro Serum-free Adrenal Competitive Binding Assay.Most preferably, the ACTH analog peptide binds to the MC-2R adrenalmembrane with at least a 2-fold greater affinity than the peptide of SEQID NO:2.

In some embodiments, the ACTH analog peptide reduces the ACTH inducedproduction of corticosterone by adrenal membrane in an in vitroSerum-free Adrenal Inhibition Assay. For example, in a fifth embodiment,ACTH analog compounds can reduce corticosteroid induction by unmodifiedACTH in explanted tissue in vitro. The ACTH analogs include peptidesthat reduce the ACTH induced production of corticosterone by adrenalmembrane in an in vitro Serum-free Adrenal Inhibition Assay.

In a sixth embodiment, extended half-life ACTH analogs are provided.Extended half-life ACTH analogs can be identified as having a firstactivity measured by the concentration of serum corticosteroid detectedin vivo that is greater than a second activity measured by theserum-free concentration of corticosteroid detected the in vitroactivity, where the in vivo activity is measured by the measured by theSerum Adrenal Corticosteroid Inhibition Assay of Example 2 and the invitro activity is measured by in vitro Serum-Free Adrenal CorticosteroidInhibition Assay of Example 4.

In a seventh embodiment, methods for screening ACTH analogs that areuseful in blocking excess ACTH while maintaining adrenal tone are alsoprovided. Various ACTH analogs can be prepared and administered to apatient to assess in vivo cortisone induction.

In an eighth embodiment, the present disclosure pertains topharmaceutical compositions comprising ACTH analogs, and theadministration thereof to a subject in a manner commensurate withtreatment for symptoms associated with an ACTH-related condition. TheACTH analogs described herein can be incorporated in pharmaceuticalcompositions for treating ACTH-related conditions, such as ACTHover-expression in humans or animals. Methods for producing ACTH analogsand associated pharmaceutical compositions containing the ACTH analogsare also provided. For example, in one aspect, methods for screening aclass of ACTH analogs for compounds useful in blocking excess ACTH whilemaintaining adrenal tone is provided.

The ACTH analog compounds can be useful in treating diseases relating tolevels of ACTH, such as conditions responsive to modulation of ACTHreceptors (such as MC-2R). Compounds useful for regulatingcorticosteroid secretion or corticosteroid levels are also provided. TheACTH analog compounds can be administered to treat conditions related tothe regulation of ACTH levels, for example to decrease the effects ofhigh levels of ACTH in patients while maintaining a tonic state ofadrenal function. The ACTH analog compositions are useful, for example,in treating ACTH-related conditions, such as Cushing's Syndrome,impaired immune response as a result of hypersecretion ofcorticosteroid, initiation of premature labor (for example, by thehypothalmus-pituitary-adrenal axis), and related conditions. In oneaspect, various ACTH analogs are prepared and administered to a patientto assess in vivo cortisone induction. In another aspect, methods fortreating veterinary subjects are provided, such as methods fordecreasing stress hormones to benefit the health of agricultural andaquacultural species grown at high population densities.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 is a graph showing the levels of corticosterone measured in vivoafter injection of various ACTH analog compounds, as compared to thelevel of corticosterone measured after administering unmodified ACTH.

FIG. 2 is a graph comparing the in vivo corticosterone induced byadministration of unmodified ACTH, an ACTH analog in combination withunmodified ACTH, the ACTH analog followed by separate administration ofthe unmodified ACTH, and the ACTH analog alone.

FIG. 3 is a graph comparing the results of a competitive binding assayfor adrenal ACTH receptors for unmodified ACTH and an ACTH analogcompound.

FIG. 4 is a graph comparing the in vitro corticosterone induced inexplanted adrenal membrane by unmodified ACTH, an ACTH analog incombination with unmodified ACTH, and the ACTH analog alone.

FIG. 5 is a graph comparing the in vivo and in vitro activity of variousACTH agonist compounds in inducing corticosterone, referenced to theactivity of unmodified ACTH (i.e., 100%).

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

As used herein, the term “unmodified Adrenocorticotropic Hormone”(“unmodified ACTH”) means the peptide hormone produced by the anteriorpituitary gland that stimulates the adrenal cortex to secreteglucocorticoid hormones, which help cells synthesize glucose through theprocess of gluconeogenesis, catabolize proteins, mobilize free fattyacids and inhibit inflammation in allergic responses. One such hormoneis corticosteroid, which regulates metabolism of carbohydrate, fat, andprotein metabolism.

The term “corticosteroid” as used herein includes the humancorticosteroid cortisol and the rodent corticosteroid corticosterone.

The term “about” used with reference to a quantity includes variationsin the recited quantity that are equivalent to the quantity recited, forinstance an amount that is insubstantially different from a recitedquantity for an intended purpose or function.

The nomenclature “P(x-y)” as used herein, where P is the name of apolypeptide and x and y are integers, refers to an amino acid sequenceconsisting of the consecutive amino acids at position (x) to position(y) of the polypeptide called “P”. For example, “hACTH(1–24)” refers tothe polypeptide of 24 consecutive amino acids consisting of the residues1 through 24 from the amine terminal end of the human ACTH peptide.Recitation of “mACTH” refers to murine ACTH. Notably, hACTH(1–24) andmACTH(1–24) are identical peptide sequences.

The nomenclature “aXb” where a and b are single-letter abbreviations foramino acids and X is a number, refers to a substitution of the aminoacid “a” in the “X” position in the unmodified ACTH peptide with theamino acid “b.” For example, “(V26F;E30K)mACTH” refers to a 39 aminoacid mouse ACTH molecule that has been modified by substituting a Pheamino acid in place of the Val amino acid at the 26 position from theamino terminus of the molecule, and substituting a Lys amino acid inplace of the Glu amino acid at the 30 position from the amino terminusof the molecule. Similarly, the nomenclature “αβχX-Yδεφ” where α, β, χ,δ, ε, and φ represent single-letter abbreviations for amino acids and Xand Y are numbers, refers to the substitution of the consecutive aminoacids “αβχ” at positions X to Y with amino acids “δεφ.”

ACTH analog compounds include compounds comprising the ACTH sequencewith one or more structural modifications that provide one or more ofthe following preferred ACTH analog biological functions: (1) reductionof corticosteroid secretion by adrenal membrane in the presence of theACTH analog compared to unmodified ACTH, (2) reduction of corticosteroidsecretion by adrenal membrane in the presence of endogenous ACTH, (3)increased MC-2R binding affinity accompanied by a reduced activation ofthe MC-2R receptor compared to unmodified ACTH. The ACTH analogcompounds are believed to act by binding to the melanocortin 2 receptor(MC-2R), weakly activating cells expressing MC-2R, and blocking theaction of endogenous ACTH on the MC-2R receptor.

Examples of preferred ACTH amino acid substitutions that can be used toform ACTH analog compounds with one or more of the desired functionsinclude: (1) substitutions of one or more amino acid residues atpositions 1–13 that conserve the amino acids at positions 6–9 and/orpromote or preserve MC-2R binding, (2) substitution of one or more aminoacid residues at positions 15–18 that prevent or antagonize enzymaticcleavage at these positions, (3) substitution of one or more amino acidresidues at positions 15–18 that are not characterized by adjacentdibasic amino acids, (4) substitution or truncation of one or more aminoacid residues at positions 20–24 that extend the serum half life of theACTH analog, (5) substitution or preferably truncation of one or moreamino acid residues at positions 25–39 that provide ACTH analogcompounds with desired release properties, or (6) truncation of aminoacid residues 25–39, with the amino acid residue at position 24 formingthe carboxy terminus of the molecule.

ACTH Analog Compounds

In a first embodiment, various ACTH analog compounds can include one ormore amino acid substitutions or truncation with respect to theunmodified human ACTH amino acid sequence. Unmodified human ACTH is apolypeptide that has 39 amino acid residues in the following sequence:N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰-Lys-Val-Tyr-Pro-Asn²⁵-Gly-Ala-Glu-Asp-Glu³⁰-Ser-Ala-Glu-Ala-Phe³⁵-Pro-Leu-Glu-Phe³⁹-Ac(SEQ ID NO: 1) (“hACTH”), where N and Ac represent the amino and carboxyterminal ends, respectively, of the molecule. ACTH(1–24) is conserved inthat the ACTH(1–24) is found in many chordates, including both humanACTH(1–24) and has the peptide sequence:N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰-Lys-Val-Tyr-Pro-Ac(SEQ ID NO: 2) (as well as being identical to the 1–24 portion of murineACTH (“mACTH(1–24)”), where N and Ac represent the amino and carboxyterminal ends, respectively, of the molecule.

ACTH analog compounds can include at least amino acid residues 1–19,more preferably amino acid residues 1–24, of hACTH (SEQ ID NO:1) withone or more amino acid substitutions. Preferred ACTH analog compoundscomprise SEQ ID NO:2 modified by one or more amino acid substitutions.ACTH analog compounds include compounds comprising the ACTH sequencewith one or more structural modifications that provide one or more ofthe following preferred ACTH analog biological functions: (1) reductionof corticosteroid secretion by adrenal membrane in the presence of theACTH analog compared to unmodified ACTH, (2) reduction of corticosteroidsecretion by adrenal membrane in the presence of endogenous ACTH and (3)increased MC-2R binding affinity with reduced activation of the MC-2Rreceptor compared to unmodified ACTH.

Particularly preferred ACTH analog compounds have one or more of thefollowing amino acid substitutions to the unmodified human ACTHsequence: (1) substitutions of one or more amino acid residues atpositions 1–13 that conserve the amino acids at positions 6–9, (2)substitution of one or more amino acid residues at positions 15–18 thatprevent or antagonize enzymatic cleavage at these positions, (3)substitution of one or more amino acid residues at positions 15–18 thatare not characterized by adjacent dibasic amino acids, (4) substitutionor truncation of one or more amino acid residues at positions 20–24 thatextend the serum half life of the ACTH analog, and (5) substitution orpreferably truncation of one or more amino acid residues at positions25–39 that provide ACTH analog compounds with extended serum half lifecompared to unmodified ACTH or compared to other ACTH analog compounds.

The ACTH analog preferably comprises a polypeptide described by theformula (I) below:N-(AA¹⁻¹³)-(AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)—Ac   (I)where N- and Ac- indicate the amino terminal end and carboxy end,respectively, of the polypeptide, (AA¹⁻¹³)- indicates a first series ofthirteen consecutive amino acids or amino acid analogs, (AA¹⁴)-represents an amino acid residue attached to the carboxy terminal end ofthe first series, (AA¹⁵⁻¹⁸)- indicates a second series of fourconsecutive amino acids attached at the carboxy terminal end of (AA¹⁴),(AA¹⁹)- represents an amino acid residue attached to the carboxyterminal end of the second series.

The ACTH analog of formula (I) preferably further comprises a portion ofa larger molecule attached to the carboxy end of the (AA¹⁹)-Ac portion.The ACTH analog can further include additional amino acids attached tothe carboxy end (Ac) of formula (I). Most preferably, ACTH analogsinclude a total of five additional amino acids attached at the Acportion of formula (I), with a total of at least 24 amino acids in theACTH analog.

ACTH analogs can comprise the amino acid sequence of unmodified ACTHcorresponding to formula (I), preferably including one or more aminoacid substitutions. Additional amino acids or amino acid analogs arepreferably attached to the carboxy terminal end of the (AA¹⁹)- residueof formula (I).

The (AA¹⁻¹³)- portion of formula (I) represents a sequence of thirteenamino acids of formula (II):-AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷-AA⁸-AA⁹-AA¹⁰-AA¹¹-AA¹²-AA¹³-   (II)having the unmodified ACTH amino acid sequence described in the secondrow of Table 1, optionally including one or more amino acidsubstitutions provided in the third row of Table 1. The (AA¹⁻¹³)-portion of unmodified human ACTH has the sequenceSer-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:3).Preferably, the AA⁶-AA⁷-AA⁸-AA⁹- portion of formula (II) has theunmodified amino acid sequence His-Phe-Arg-Trp- (SEQ ID NO:4),optionally substituted with one or more (D) amino acid analogs thereof.

TABLE 1 ACTH analog amino acid substitution of (AA^(1–13)) Residue: AA¹-AA²- AA³- AA⁴- AA⁵- AA⁶- AA⁷- AA⁸- AA⁹- AA¹⁰ AA¹¹- AA¹²- AA¹³-Unmodified Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val ACTHResidue: Optional (D)Ser (D)Tyr (D)Ser (D)Met (D)Glu (D)His (D)Phe(D)Arg (D)Trp Cys (D)Lys (D)Pro (D)Val ACTH analog Nle Cys (D)-p-iodoPheLys Gly Amino Acid Asp (D)-l-naphAla Om Substitutions Dab Dpr

In Table 1, the designation (D) refers to the (D) enantiomer of theindicated amino acids; Nle refers to the amino acid analog norleucine oranother amino acid with an alkyl side chain; Orn refers to ornithine, oranother modified amino acid with a similar side chain; Dab refers to 2,4diaminobutyric acid, or a similar diamino acid; Dpr refers to 2,3diaminopropionic acid or another diamino acid, (D) p-iodo-Phe refers tosteric group such as a p-iodo modified Phe amino acid; and(D)-I-naph-Ala refers to the I-naph- modified (D)-Ala amino acid, oranother sterically modified amino acid. ACTH analog compounds thatinclude substitutions to the unmodified (AA¹⁻¹³) portion of theunmodified ACTH preferably include one of the optional ACTH analog AminoAcid residues indicated below a given residue position in Table 1. Forexample, unmodified ACTH has a Glu residue at the AA⁵ position, that canoptionally be substituted with a (D)-Glu, Cys or Asp residue in formingan ACTH analog. The amino acid sequence of formula (II) can also includeone or more amino acid substitutions corresponding to MC-2R bindingsequences disclosed by Hruby et al. in U.S. Pat. Nos. 4,485,039;4,457,864; 4,866,038; 5,731,408; 5,714,576; 5,049,547; 4,918,055;4,649,191; and 5,674,839, which are incorporated herein by reference.

The (AA¹⁴)- portion of formula (I) represents an amino acid residue thatis preferably Gly or (D) Gly, although other amino acids can besubstituted at the (AA¹⁴)- position that preserve desirable biologicalfunctions of ACTH analog compounds, such as the reduction ofcorticosteroid secretion by the ACTH analog compared to ACTH, reductionof corticosteroid secretion by endogenous ACTH and/or MC-2R binding.

The (AA¹⁵⁻¹⁸)- portion of formula (I) represents a sequence of fouramino acids of formula (III):-AA¹⁵-AA¹⁶-AA¹⁷-AA¹⁸-.   (III)The (AA¹⁵⁻¹⁸)- portion of unmodified human ACTH comprises the sequenceLys-Lys-Arg-Arg (SEQ ID NO:5), which includes four adjacent amino acidswith basic side chains. Preferably, ACTH analog compounds includesubstitution of one or more amino acid residues in formula (III) thatprevent or antagonize enzyme cleavage at these positions. Alsopreferably, ACTH analog compounds include substitution of one or moreamino acid in formula (III) that are not characterized by adjacent aminoacids with basic side chains. AA¹⁵- and AA¹⁶- are preferablyindependently selected from the group consisting of: Lys, Ala, Gly andVal; AA¹⁷-AA¹⁸ are preferably independently selected from the groupconsisting of: Arg, Ala, Gly and Val.

In one aspect, one or more amino acid substitutions of residuesAA¹⁶-AA¹⁷-AA¹⁸ of SEQ ID NO:2 or formula (III) are selected such thatthe amino acid residues 16, 17 and 18 of the ACTH analog do not includeany two adjacent amino acid residues selected from the group consistingof: Lys and Arg. In other words, in some ACTH analogs, the amino acidsLys and Arg do not appear adjacent to each other (i.e., an arginineadjacent to a lysine or vice versa) or to themselves (i.e., an arginineadjacent to another arginine, or a lysine adjacent to another lysine)within the AA¹⁶-AA¹⁷-AA¹⁸ portion of the peptide sequence. Instead, oneor more of the amino acids within the AA¹⁶-AA¹⁷-AA¹⁸ portion aresubstituted with any amino acid or amino acid analog other than Lys orArg that provide one or more of the following preferred ACTH analogbiological functions: (1) reduction of corticosteroid secretion byadrenal membrane in the presence of the ACTH analog compared tounmodified ACTH, (2) reduction of corticosteroid secretion by adrenalmembrane in the presence of endogenous ACTH and (3) increased MC-2Rbinding affinity with reduced activation of the MC-2R receptor comparedto unmodified ACTH. For example, ACTH analog compounds can include Alaresidues substituted for the unmodified ACTH residues at one or morepositions in formula (III). ACTH analog compounds can include residuesubstitution of SEQ ID NO: 5 in formula (III) of Gly, or any amino acidwith an alkyl side chain (i.e., Ala, Val, Leu, Ile, or an amino acidanalog comprising an alkyl side chain such as Nle). Also preferably, theACTH analog can also include at least one Ala and at least one Argresidue substituted at any two of the amino acid positions 15, 16, 17 or18 of SEQ ID NO:2. Optionally, the ACTH analog substitutions atpositions 15, 16, 17, or 18 of SEQ ID NO:2 are amino acids selected fromthe group consisting of: Lys, Arg, Ala, Gly, Val, Leu, lie, an aminoacid analog comprising an alkyl side chain such as Nle, Gln, Asn, Glu,and Asp. Most preferably, the ACTH analogs comprise an amino acidsequence according to formula (III) selected from the group consistingof: Lys-Arg-Ala-Ala- (SEQ ID NO:6), Ala-Lys-Ala-Arg (SEQ ID NO:7),Lys-Ala-Ala-Arg (SEQ ID NO:8), Lys-Ala-Arg-Ala- (SEQ ID NO:9),Gln-Lys-Gln-Arg (SEQ ID NO:10) and Ala-Ala-Ala-Ala (SEQ ID NO:11). ACTHanalog compounds can also include the substitution of one or more aminoacid residues at positions 15, 16, 17 or 18 with an amino acid or aminoacid analog having an alkyl side chain, including Gly, Ala, Val, Leu,lie or NIe. ACTH analogs include other amino acids substituted at one ormore of the AA¹⁵-AA¹⁶-AA¹⁷-AA¹⁸ positions that preserve one or morepreferred biological functions of ACTH analog compounds, such as thereduction of corticosteroid secretion by the ACTH analog compared tounmodified ACTH, reduction of corticosteroid secretion by endogenousACTH and/or MC-2R binding.

The (AA¹⁹)- portion of formula (I) represents an amino acid residue thatis can be Pro, Trp or Ala, but is preferably Trp, Ala or other aminoacids that can be substituted at the (AA¹⁹)- position while preservingone or more preferred biological functions of ACTH analog compounds,such as the reduction of corticosteroid secretion by the ACTH analogcompared to unmodified ACTH, reduction of corticosteroid secretion byendogenous ACTH and/or MC-2R binding. Preferably, the ACTH analogcompounds have an amino acid other than Proline substituted at the(AA¹⁹)- position. More preferably, the ACTH analogs include the aminoacid Trp substituted at the (AA¹⁹)- position, instead of Pro. UnmodifiedACTH peptide sequences, such as hACTH and mACTH, comprise a prolineresidue at the (AA¹⁹)- position. Proline has the following chemicalstructure:

The chemical structure of Proline includes a side-chain that forms aring structure. Proline is often found at the end of a helix or in turnsor loops. Unlike other amino acids which exist almost exclusively in thetrans-form in polypeptides, proline can exist in the cis-configurationin peptides. The cis and trans forms are nearly isoenergetic.Preferably, (AA¹⁹)- is Trp in ACTH analogs comprising SEQ ID NOs:6–10 atformula (III), although compounds where (AA¹⁹)- is Pro in ACTH analogsare also provided.

Accordingly, particularly preferred ACTH analogs are modified hACTH,mACTH or hACTH(1–24) peptide sequences where (AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)-are selected from the group consisting of:-Gly¹⁴-Lys¹⁵-Arg¹⁶-Ala¹⁷-Ala¹⁸-Trp¹⁹- (SEQ ID NO:12);-Gly¹⁴-Ala¹⁵-Lys¹⁶-Ala¹⁷-Arg¹⁸-Pro¹⁹- (SEQ ID NO:13);-Gly¹⁴-Lys¹⁵-Ala¹⁶-Ala¹⁷-Arg¹⁸-Pro¹⁹- (SEQ ID NO:14);-Gly¹⁴-Lys¹⁵-Ala¹⁶-Arg¹⁷-Ala¹⁸-Pro¹⁹- (SEQ ID NO:15);-Gly¹⁴-Gln¹⁵-Lys¹⁶-Gln¹⁷-Arg¹⁸-Pro¹⁹- (SEQ ID NO:16) and-Gly¹⁴-Lys¹⁵-Arg¹⁶-Ala¹⁷-Ala¹⁸-Pro¹⁹- (SEQ ID NO:17).

The ACTH analog is comprising a polypeptide described by formula (IVa)or formula (IVb):N-(AA¹⁻¹³)-(AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)-(AA²⁰⁻²⁴)-   (IVa)N-(AA¹⁻¹³)-(AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)-(AA²⁰⁻²⁴)-Ac   (IVb)where N- indicates the N-terminal end of the polypeptide, Ac indicatesthe carboxy terminal end of the polypeptide. The ACTH analogs of formula(VIa) or (VIb) include the N-(AA¹⁻¹³)-(AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)- asdescribed with respect to formula (I) above, and further comprise the(AA²⁰⁻²⁴)- portion attached to the carboxy end of the amino acidsequence of formula (I). The (AA²⁰⁻²⁴)- portion of formula (VIa) and(VIb) represents a sequence of five amino acids of formula (V):-AA²-AA²¹AA²²AA²³-A²⁴.   (V)The (AA²⁰⁻²⁴)- portion of unmodified human ACTH comprises the sequenceVal-Lys-Val-Tyr-Pro (SEQ ID NO:18). Preferably, ACTH analog compoundsinclude substitution of one or more amino acid residues in formula (V)extend the serum half life of the ACTH analog. For example, an ACTHanalog can comprise a sequence corresponding to formula (V) of:-Ala-Ala-Ala-Ala-Ala- (SEQ ID NO:19).

Particularly preferred ACTH analog according to formula (IVa) comprisethe sequence hACTH(1–24) with substitution of the -AA¹⁵-AA¹⁶-AA¹⁷-AA¹⁸-portion of formula (III) with SEQ ID NO:6. One particularly preferredcompound of formula (IVa) is the ACTH analog (KKRRP15-19KRAAW)mACTH(1–24), having the sequence:N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Arg-Ala-Ala-Trp-Val²⁰-Lys-Val-Tyr-Pro-Ac(SEQ ID NO:20), also referred to as “AT814”. Other particularlypreferred ACTH analog compounds consist essentially of the peptide ofSEQ ID NO:2 with alanine or glutamine substitutions of one or more ofthe amino acid residues at positions 15, 16, 17 or 18, and/orsubstitution of the amino acid residues at positions 20, 21, 22, 23 or24 with alanine, for example as described by Costa, J L et al.,“Mutational analysis of evolutionarily conserved ACTH residues,” GenComp Endocrinol. March;136(1):12–6 (2004), which is incorporated hereinby reference in its entirety.

In a third aspect of the first embodiment, the ACTH analog is comprisinga polypeptide described by formula (VIa) or formula (VIb):N-(AA¹⁻¹³)-(AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)-(AA²⁰⁻²⁴)-(AA²⁵⁻³⁹)-   (VIa)N-(AA¹⁻¹³)-(AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)-(AA²⁰⁻²⁴)-(AA²⁵⁻³⁹)-Ac   (VIb)where N- indicates the amino-terminal end of the polypeptide, Acindicates the carboxy-terminal end of the polypeptide. The ACTH analogsof formula (IVa) or (IVb) include theN-(AA¹⁻¹³)-(AA¹⁴)-(AA¹⁵⁻¹⁸)-(AA¹⁹)-(AA²⁰⁻²⁴)- as described above withrespect to formula (IVa), and further comprise the (AA²⁵⁻³⁹)- portionattached to the carboxy end of the amino acid sequence of formula (IVa).Formula (VIa) can optionally include additional chemical structuresattached to the AA³⁹ residue, while the AA³⁹ residue forms the carboxyterminal end of the structures of formula (VIb). The (AA²⁵⁻³⁹)- portionof formula (VIa) and (VIb) represents a sequence of fifteen amino acidsof formula (VII):-AA²⁵-AA²⁶-AA²⁷-AA²⁸-AA²⁹-AA³⁰-AA³¹AA³²-A³³-AA³⁴-AA³⁵-AA³⁶-AA³⁷-AA³⁸-AA³⁹.  (VII)

The (AA²⁵⁻³⁹)- portion of unmodified human ACTH comprises the sequenceAsn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe (SEQ IDNO:21). Preferably, ACTH analog compounds include substitution of one ormore amino acid residues in formula (VII), or truncation of one or moreamino acid residues from the carboxy-terminal end of formula (VII), thatprovide ACTH analog compounds with desired sustained release properties.For example, an ACTH analog can comprise a sequence corresponding toformula (VII) having the unmodified ACTH sequence of SEQ ID NO:10,optionally modified by substitution of Lys at AA³⁰ and/or substitutionof Arg at AA³⁶.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Such substitutions may be made in accordance with thefollowing Table 2 when it is desired to finely modulate thecharacteristics of the protein. Table 2 shows amino acids which may besubstituted for an original amino acid in a protein and which areregarded in the art as conservative substitutions. ACTH analog compoundsinclude compounds with one or more conservative substitutions thatretain one or more of the following preferred ACTH analog biologicalfunctions: (1) reduction of corticosteroid secretion by adrenal membranein the presence of the ACTH analog compared to unmodified ACTH, (2)reduction of corticosteroid secretion by adrenal membrane in thepresence of endogenous ACTH and (3) increased MC-2R binding affinitywith reduced activation of the MC-2R receptor compared to unmodifiedACTH.

TABLE 2 Original Conservative Residue Substitutions Ala ser Arg lys Asngln, his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu,val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than in Table 2,i.e., selecting residues that differ more significantly in their effecton maintaining: (a) the structure of the polypeptide backbone in thearea of the substitution, for example, as a sheet or helicalconformation; (b) the charge or hydrophobicity of the molecule at thetarget site; or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in proteinproperties will be those in which: (a) a hydrophilic residue, e.g.,seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteineor proline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histadyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

The preferred ACTH analog biological functions of ACTH analog compoundscan be measured by any suitable method, but are preferably assessed byperforming one or more of the assays described below

ACTH Analog Compounds with Decreased ACTH Function

In a second embodiment, ACTH analogs are modified ACTH peptides thatfunction to reduce corticosteroid secretion by adrenal membrane in thepresence of the ACTH analog compared to unmodified ACTH. The structureof the ACTH analogs is preferably selected according to one or more ofthe structural formulae provided above. The ACTH analog compounds canhave a reduced ACTH-mediated secretion of blood corticosterone, forexample as measured by a reduction in the level of corticosteroid in theblood of a subject after administering the ACTH analog. ACTH analogspreferably demonstrate at least a 10%, 15%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%reduction in the serum corticosterone levels compared to the comparableadministration of unmodified ACTH peptide, as measured by an in vivoSerum Corticosteroid Induction Assay.

An in vivo Serum Corticosteroid Induction Assay measures the level ofcorticosteroid or corticosterone in the blood stream of a subject. Morespecifically, the in vivo Serum Corticosteroid Induction Assay caninclude the following steps: first, an agent to suppress endogenous ACTHproduction was administered to a mouse subject, such as anintraperitoneal injection of dexamethasone; second, after waiting asuitable period of time for suppression of endogenous ACTH production(e.g., 1.5–2.0 hours), a test compound can be administered to the mouseby a suitable method, such as intraperitoneal or subcutaneous injection;and third, after waiting a suitable period of time (e.g., 1 hour) foraction of the injected test compound, a blood sample can be taken fromthe subject and the level of serum corticosterone can be determined by asuitable method, such as a competitive radioimmunoassay using ¹²⁵I RIAkit (ICN, Costa Mesa, Calif.). Example 1 describes a murine in vivoSerum Corticosteroid Induction Assay in detail.

The corticosteroid or corticosterone secretion induced in vivo by anACTH analog compound was compared to the level of corticosteroidsecretion produced by an unmodified ACTH compound by measuring bloodserum corticosteroid after administering either the ACTH or ACTH analogas test compounds. FIG. 1 shows the results of murine in vivoCorticosteroid Adrenal Induction Assays performed with mACTH(1–24) andvarious ACTH analog test compounds according to the method of Example 1.ACTH analog compositions with decreased function were identified bymeasuring corticosterone levels using standard radioimmunoassay (RIA)analysis on blood samples collected one hour after administering themACTH(1–24) or ACTH analog compound to dexamethasone-suppressed mice. InFIG. 1, the potency of various ACTH analog peptides is expressed as percent induction of corticosterone, with mouse ACTH being 100%. The graph10 shows the percent corticosterone induction 12 for ACTH and ninesamples 14 of ACTH analog Compounds. The percent of corticosteroneinduction 12 of ACTH analog Compounds in Samples 2–10 are shown as apercentage of the concentration of serum corticosterone 20 measured forunmodified mACTH(1–24) in the in vivo Serum Corticosteroid AdrenalInduction Assay. The structural modifications and measured percentagereduction in serum measured for Samples 2–10 in FIG. 1 are shown inTable 3 below.

TABLE 3 Percent reduction Label in in serum Sample FIG. 1 StructuralModification corticosteroid 2 20 (V26F, E30K)mACTH 18.3% 3 25 (P19W,K21E, Y23R)mACTH 36.5% 4 30 (E30K, P36R)mACTH 41.5% 5 35(PVKVYP19-24AAAAA)mACTH   51% 6 40 (V26F, P36R)mACTH 59.3% 7 45 (P19W,K21E)mACTH 61.1% 8 50 (P19W, K21A, delY23)mACTH 76.0% 9 55 (P19W,K21A)mACTH 83.4% 10 60 (KKRRP15-19KRAAW)mACTH  100%Reduction of Endogenous ACTH-Induced Corticosterone Induction

In a third embodiment, ACTH analogs are modified ACTH peptides thatfunction to reduce corticosteroid secretion by adrenal membrane in thepresence of endogenous ACTH. When administered before or concurrentlywith ACTH, ACTH analog compounds can produce a 10–100% reduction inserum corticosteroid levels. Preferably, ACTH analog compounds provide areduction in the serum corticosteroid levels measured by an in vivoSerum Corticosteroid Inhibition Assay of about 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% compared to administration of ACTH alone, as measured by anin vivo Serum Corticosteroid Inhibition Assay, such as the assaydescribed in Example 2. Exemplary ACTH analog compounds that inhibitadrenal hormone production induced by endogenous ACTH were identified byperforming a series of in vivo Serum Corticosteroid Inhibition Assays.ACTH analogs can be administered alone, prior to ACTH administration, orconcurrently in combination with ACTH.

An in vivo Serum Corticosteroid Inhibition Assay measures the level ofcorticosteroid or corticosterone in the blood stream of a subject afteradministering a compound having a first corticosteroid-producingbiological activity and a test compound. The compound having thecorticosteroid-producing biological activity can be any compound knownto induce a detectable increase in corticosteroid production. The testcompound can be administered before the corticosteroid-producingcompound, in combination with the corticosteroid-producing compound,and/or after the corticosteroid-producing compound. Thecorticosteroid-producing compound is preferably ACTH, but can also be anACTH analog with a known corticosteroid-producing biological activity.By performing a series of in vivo Serum Corticosteroid Inhibition Assaysusing ACTH analog test compounds, ACTH analogs that inhibit theproduction of corticosteroid relative to the corticosteroid-producingcompound used can be identified. Preferably, the ACTH analogs identifiedin this manner inhibit the production of corticosteroid in the presenceof endogenous unmodified ACTH.

The in vivo Serum Corticosteroid Inhibition Assay can include sequentialadministration of the test compound and the corticosteroid-producingcompound by the following steps: first, an agent to suppress endogenousACTH production was administered to a mouse subject, such as anintraperitoneal injection of dexamethasone; second, after waiting asuitable period of time for suppression of endogenous ACTH production(e.g., 1.5–2.0 hours), a test compound was administered to the mouse bya suitable method, such as intraperitoneal injection; third, afterwaiting a suitable period of time (e.g., 1 hour) for action of the testcompound, a compound with corticosteroid-producing activity can beadministered to the mouse by a suitable method, such as intraperitonealinjection; and fourth, after waiting a suitable period of time for thebiological activity of both administered compounds to occur (e.g., 1hour), a blood sample was taken from the subject and the level of serumcorticosterone was determined by a suitable method, such as acompetitive radioimmunoassay using ¹²⁵I RIA kit (ICN, Costa Mesa,Calif.). Optionally, steps two and three can be reversed (administeringthe corticosteroid-producing compound first, followed by administrationof the test compound). The in vivo Serum Corticosteroid Inhibition Assaycan also include co-administration of the test compound and thecorticosteroid-producing compound by the following steps: first, anagent to suppress endogenous ACTH production was administered to a mousesubject, such as an intraperitoneal injection of dexamethasone; second,after waiting a suitable period of time for suppression of endogenousACTH production (e.g., 1.5–2.0 hours), the corticosteroid-producingcompound and a test compound were co-administered to the mouse by asuitable method, such as intraperitoneal injection; and third, afterwaiting a suitable period of time for the biological activity of bothadministered compounds to occur (e.g., 1 hour), a blood sample was takenfrom the subject and the level of serum corticosterone was determined bya suitable method, such as a competitive radioimmunoassay using ¹²⁵I RIAkit (ICN, Costa Mesa, Calif.). Also, in some embodiments, the first stepin any in vivo Serum Corticosteroid Inhibition Assay can be replaced bya measurement of the initial endogenous corticosteroid or corticosteroidlevel in the blood of the subject in the manner of step four, instead ofadministering the agent to suppress ACTH production. Example 2 describesmurine in vivo Serum Corticosteroid Inhibition Assays in detail.

A series of separate in vivo Serum Corticosteroid Inhibition Assays wereperformed to identify ACTH analogs that inhibit ACTH adrenal hormoneproduction in vivo. The serum corticosterone levels ofdexamethasone-suppressed mice were measured as described in Example 2after administration of (1) ACTH (corticosteroid-producing compound),(2) an ACTH analog (test compound) followed by subsequent administrationof ACTH, (3) a combination of ACTH and the ACTH analog and (4) the ACTHanalog alone. Table 4 shows the level of serum corticosteroid measuredin the mice as a function of time of administration.

TABLE 4 Combined administration of ACTH and an ACTH analog Time Sample 0minutes 90 minutes 120 minutes 180 minutes Vehicle Dexamethasone Vehicle0 ± 0 ng/mL ACTH (administered mACTH(1–24) 500 ± 76 ng/mL AT90 − ACTH120to all groups) (KKRRP15-19KRAAW) mACTH(1–24) 175 ± 27 ng/mL mACTH(1–24)(AT + ACTH)120 (KKRRP15-19KRAAW) 51 ± 8 ng/mL mACTH(1–24) + mACTH(1–24)AT (KKRRP15-19KRAAW) 7 ± 7 ng/mL mACTH(1–24)

Referring to Table 4, dexamethasone was first administered at time=0minutes to mice in five separate in vivo Serum Corticosteroid Assays, asdescribed in Example 2. In the “Vehicle” sample, the liquid vehiclealone was administered 120 minutes after dexamethasone injection,resulting in an undetectable level of corticosterone measured in a bloodsample taken one hour later. In the “ACTH” sample, ACTH was administered120 minutes after dexamethasone injection, resulting in a level of500±76 ng/mL of corticosterone detected in a blood sample taken one hourlater. In the “AT90-ACTH120” sample, the ACTH analog (KKRRP15-19KRMW)mACTH(1–24) was administered at 1.5 hours after dexamethasone injection,ACTH(1–24) was administered 30 minutes later, and a serum corticosteronelevel of 175±27 ng/mL was measured in a blood level taken one hourlater. In the “(AT+ACTH)120” sample, the ACTH analog (KKRRP15-19KRAAW)mACTH(1–24) (“AT814”) was administered together with mACTH(1–24) 120minutes after dexamethasone injection, and a serum corticosterone levelof 51±8 ng/mL was measured in a blood level taken one hour later. In the“AT” sample, the administration of the ACTH analog (KKRRP15-19KRAAW)mACTH(1–24) 120 minutes after dexamethasone injection resulted in anegligible level of serum corticosterone level of about 7±7 ng/mL.

FIG. 2 shows the results from Table 2 as a percentage of the level ofcorticosterone levels measured at 180 minutes after dexamethasoneinjection, as described in Example 2. The graph 100 shows the percentcorticosterone induction 112 for ACTH and four samples 114 described inTable 2, with the level of corticosterone measured for administration ofthe ACTH 120 being normalized to 100%. Sample “AT90-ACTH120” 140 showsthe administration of the ACTH analog 30 minutes before administrationof the ACTH, which resulted in about a 65% reduction in serumcorticosterone levels in blood samples taken one hour after ACTHadministration. Sample “(AT+ACTH)120” 160 shows the concurrentadministration of the ACTH analog with the ACTH and resulted in about a90% reduction in serum corticosterone levels in blood samples taken onehour later. As seen in the “AT” Sample 180, as well as Sample 10 ofTable 1, the administration of the ACTH analog alone again resulted inabout a 99%–100% reduction in serum corticosterone levels in bloodsamples taken one hour later. The “Vehicle” only sample is not shown inFIG. 2.

Adrenal ACTH Receptor Binding Assay

In a fourth embodiment, ACTH analogs are modified ACTH peptides thatfunction to bind to adrenal ACTH receptors, such as the MC-2R receptor,preferably with increased MC-2R binding affinity and reduced activationof the MC-2R receptor compared to unmodified ACTH.

Preferred ACTH analog compounds bind to adrenal membranes with a greateraffinity than endogenous unmodified ACTH, and are able to displaceunmodified ACTH in an in vitro Serum-free Adrenal Competitive BindingAssay (Example 3). ACTH analog compounds preferably displace at least20% of ACTH binding to adrenal membrane preparations as measured by thein vitro Serum-free Adrenal Competitive Binding Assay. More preferably,ACTH analog compounds can have a 2-, 3-, 4-, 5-, 10-, 20-, 30-, 40-,50-, 60-, 70-, 80-, 90-, 100-fold greater affinity for explanted adrenalmembrane compared to unmodified ACTH.

ACTH analog compounds that bind to adrenal membrane with a greateraffinity than endogenous unmodified ACTH, and are able to displaceunmodified ACTH can be identified using an in vitro Serum-free AdrenalCompetitive Binding Assay (Example 3). FIG. 3 shows the results of an invitro Serum-free Adrenal Competitive Binding Assay performed by themethod described in Example 3 using the exemplary ACTH analog AT814,having SEQ ID NO:20 (“AT814”). The graph 200 shows the counts per minute212 (CPM) measured by a gamma counter in adrenal membrane preparationsincubated with radioactive ACTH. A higher CPM level indicates thepresence of greater amounts of the radiolabelled compound. Column 210shows the background measurement without any adrenal membrane present.Column 220 indicates the level of radio-labeled unmodified mACTH(1–24)detected after combining the explanted adrenal membrane with only theradio-labeled mACTH(1–24) for 2 hours.(no competitive binding). Columns230, 240, and 250 show the results of competitive binding assays forbinding of non-radio-labeled (“cold”) mACTH(1–24) to displace theradio-labeled mACTH(1–24) from the explanted adrenal membrane (measuredin column 220) by competitive binding to receptors such as MC-2R on theadrenal membrane. Specifically, column 230 indicates a decrease in thelevel of radio-labeled mACTH(1–24) detected after combining theradio-labeled explanted adrenal membrane with 10 nM “cold” mACTH(1–24);column 240 indicates additional reduction in the level of radio-labelledmACTH(1–24) bound to the radio-labeled explanted adrenal membrane afteradding 100 nM “cold” mACTH(1–24); and Column 250 indicates furtherreduction in the level of radio-labelled mACTH(1–24) attached to theradio-labeled explanted adrenal membrane after combining an explantedadrenal membrane preparation with 1000 nM of “cold” mACTH(1–24). Thedecreasing levels of radio-labeled mACTH(1–24) detected as theconcentration of “cold” mACTH(1–24) is increased indicates adisplacement of the radio-labeled mACTH(1–24) by the “cold” mACTH(1–24)from the MC-2R receptor.

The in vitro Serum-free Adrenal Competitive Binding Assay was repeatedusing the “cold” (non-radio-labeled) AT814 ACTH analog compounddescribed by SEQ ID NO:20, instead of the mACTH(1–24). Columns 235, 245,and 255 show the results of competitive binding assays for binding ofnon-radio-labeled AT814 to displace the radio-labeled mACTH(1–24) fromthe explanted adrenal membrane (measured in column 220) by competitivebinding of “cold” AT814 at concentrations of 10 nM (column 235), 100 nM(column 245) and 1000 nM (column 255). The decreasing levels ofradio-labeled AT814 detected as the concentration of “cold” AT814 isincreased indicates a displacement of the radio-labeled mACTH(1–24) bythe “cold” AT814. Specifically, the results in graph 200 indicate thatAT814 has an increased affinity to bind to the adrenal membrane that isabout 3- to about 4-fold greater than the radio-labeled mACTH(1–24). Theincreased affinity of AT814 binding to adrenal membrane can beindicative of increased MC-2R receptor binding affinity.

Testing of ACTH Analog Compounds In Vitro

In a fifth embodiment, ACTH analog compounds can reduce corticosteroneinduction by unmodified ACTH in explanted tissue in vitro. When combinedwith explanted adrenal membrane ACTH analog compounds preferably producea 10–100% reduction in corticosteroid levels in serum-free media.Preferably, ACTH analog compounds provide a reduction in the serumcorticosteroid levels measured by an in vitro Serum-Free AdrenalCorticosteroid Inhibition Assay of about 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% compared to administration of unmodified ACTH alone (Example 4).Exemplary ACTH analog compounds that inhibit adrenal hormone productioninduced by exposing explanted adrenal membrane to unmodified ACTH wereidentified by performing a series of in vitro Serum-Free AdrenalCorticosterone Inhibition Assays, suggesting a direct action of ACTHanalog compounds on adrenal corticosterone production.

A series of separate in vitro Serum-Free Adrenal CorticosteroidInhibition Assay were performed to identify ACTH analogs that inhibitACTH-induced adrenal hormone production in vitro. The concentration ofcorticosterone was measured according to the procedure of Example 4 in aserum-free culture medium containing explanted adrenal membrane andunmodified ACTH, AT814 ACTH analog (SEQ ID NO:20), or a combination ofACTH and AT814 at 100 ng/mL each. Table 3 shows the level ofcorticosteroid measured in the serum free medium in a series of in vitroSerum-Free Adrenal Corticosterone Inhibition Assays performed usingexplanted mouse adrenal membrane. Corticosterone levels were taken at2.0 hours after the adrenal membrane was placed in the serum free M199medium, and each corticosterone level in Table 5 is an average of fivesamples.

TABLE 5 Combined administration of ACTH and an ACTH analog Sample 0 hrs.0.5 hrs. 2.5 hrs. M199 Medium Incubate tissue 278 mg/mL ACTH in M199serum- ACTH 600 mg/mL AT-814 free medium AT814 290 mg/mL ACTH + AT814(applied to all ACTH + AT814 396 mg/mL samples)

Referring to Table 5, explanted mouse adrenal halves were placed in anM199 serum free medium (Invitrogen) for 30 minutes, as provided inExample 4. In the “M199 Medium” samples, the adrenal membrane was soakedin the M199 serum-free medium for a total of 2.0 hours after the initial30 minutes (2.5 hours total), before the corticosterone level in theculture was measured using standard RIA radioassay techniques. In the“ACTH” samples, unmodified ACTH was added to the M199 medium, resultingin a level of about 600 mg/mL of corticosterone detected in the culturemedium two hours later. In the “AT-814” sample, the ACTH analog(KKRRP15-19KRAAW) mACTH(1–24) (SEQ ID NO:20) was added to the M199medium, resulting in a level of about 290 mg/mL of corticosteronedetected in the culture medium two hours later. In the “ACTH+AT-814”sample, 100 ng/mL of AT814 ACTH analog (SEQ ID NO:20) and 100 ng/mL ofunmodified ACTH was added together to the M199 medium, resulting in alevel of about 396 mg/mL of corticosterone detected in the culturemedium two hours later. FIG. 4 is a graph 300 showing the percentcorticosterone induction 302 from Table 3 as a percentage relative tothe amount of ACTH 310 measured in the M199 culture medium for the“ACTH” sample 310, the “AT-814” sample 320 and the “ACTH+AT-814” sample330, as described above and in Example 4. The “M199 Medium” sample isnot shown in FIG. 2. Referring again to FIG. 4, the addition of theAT814 to the adrenal membrane in the M199 serum-free medium permittedonly a negligible amount of corticosterone induction, and addition ofthe combination of equal amounts of the AT814 ACTH analog and unmodifiedACTH reduced the amount of corticosterone induction by about 65%compared to addition of the unmodified ACTH alone.

Testing of ACTH Analog Compounds in vivo

ACTH analog compounds with extended serum half lives can be preferredfor certain applications. ACTH analog compounds can optionally includeone or more amino acid substitutions or truncations that can extend theserum half life of the ACTH analog relative to unmodified ACTH oranother ACTH analog. For example, ACTH analog compounds can comprise oneor more amino acid substitutions from the amino acid positions 25–39 ofan unmodified ACTH sequence that that extend the serum half life of thecompound relative to the ACTH sequence. Preferred ACTH analog compoundscan have a serum half life that is greater than that of unmodified ACTH.The half life of ACTH in blood is less than about 20 minutes. Therefore,particularly preferred ACTH analog compounds have a serum half life ofabout 20, 30, 40, or 50 minutes or greater.

In a sixth embodiment, extended half-life ACTH analogs are provided.Extended half-life ACTH analogs can be identified as having a firstactivity measured by the concentration of serum corticosteroid detectedin vivo that is greater than a second activity measured by theserum-free concentration of corticosteroid detected in the in vitroactivity, where the in vivo activity is measured by the Serum AdrenalCorticosteroid Inhibition Assay of Example 1 and the in vitro activityis measured by in vitro Serum-Free Adrenal Corticosteroid InhibitionAssay of Example 4.

While amino acid substitutions at other positions may also enhance ACTHanalog serum half life in some circumstances, certain embodimentsprovide for extending the serum half life of an ACTH analog by modifyingone or more amino acids at the 20–24 positions of an ACTH molecule,including hACTH or mACTH. FIG. 5 is a graph comparing the in vivoactivity (grey bars) and in vitro activity (white bars) of seven ACTHanalog compounds, 410, 420, 430, 440, 450, 460, and 470. For all sevenACTH analog compounds, the in vivo activity was measured using themethod of Example 1 and the in vitro activity was measured using themethod of Example 4. The results are provided as a bar graph of the “%corticosterone induction” 402 measured as a percentage relative to themACTH(1–24) sequence (SEQ ID NO:2). As seen in FIG. 5, sample 460,designated “Ala19–24” refers to the ACTH analog(AAAAA20-24VKVYP)ACTH(1–24), comprising a substitution of Ala residuesat each of the amino acid 19–24 positions of mACTH. Sample 460 showed anincreased serum half life measured in vivo 462 relative to both the invitro activity 464 measured for this compound, as well as being about50% greater than the in vitro activity of unmodified mACTH. Furthermore,the in vivo activity of the (AAAAA20-24VKVYP)ACTH(1–24) ACTH analog 460was greater relative to both mACTH and to other ACTH analogs 410, 420,430, 440, 450 and 470. These results suggest that the amino acidresidues at the 20–24 positions may play a role in determining the serumhalf life of an ACTH analog compound. ACTH analog compounds with one ormore amino acid substitutions at the 20–24 positions of ACTH areparticularly preferred for extending the serum half life relative toACTH.

Also desirably, ACTH analog compounds can include amino acidsubstitutions that extend the serum half life without appreciablyincreasing, or more preferably decreasing, the corticosterone inductionof the ACTH analog, as measured by in vivo (Example 1) or in vitro(Example 4) Assays. For example, corticosterone induction fromsubstitutions of the 15–19 positions in ACTH analogs 410, 420, 430, 440,450 and 470 reduced both the in vivo and in vitro corticosteroneinduction activity of the ACTH analogs compared to unmodifiedmACTH(1–24). Specifically, ACTH analog 410 is a (K15A, R17A)mACTH(1–24)ACTH analog that demonstrated an in vitro activity 414 that was greaterthan its in vivo activity 412; ACTH analog 420 is a (K16A,R17A)mACTH(1–24) ACTH analog that demonstrated an in vitro activity 424that was greater than its in vivo activity 422; ACTH analog 430 is a(K16A, R18A)mACTH(1–24) ACTH analog that demonstrated an in vitroactivity 434 that was greater than its in vivo activity 432; ACTH analog440 is a (K15Q, R17Q)mACTH(1–24) ACTH analog that demonstrated an invitro activity 444 that was greater than its in vivo activity 442 andACTH analog 470 is a (P19W, K21A)mACTH(1–24) ACTH analog thatdemonstrated an in vitro activity 474 that was greater than its in vivoactivity.

Screening Methods

In a seventh embodiment, methods for screening ACTH analogs that areuseful in blocking excess ACTH while maintaining adrenal tone are alsoprovided. Various ACTH analogs can be prepared and administered to apatient to assess in vivo cortisone induction.

The specificity of molecular recognition of certain compounds (i.e.,antigens by antibodies) to form a stable complex can serve as the basisof both the analytical immunoassay in solution and the immunosensor onsolid-state interfaces. These analytical methods can be ligand-bindingassays based on the observation of the products of the ligand-bindingreaction between the target analyte (i.e., a compound that binds to MC2Rreceptors) and a highly specific binding reagent.

In certain instances, alternative analyte-binding compounds such asaptamers are applied to ligand-binding assays for particularly sensitivescreening. Aptamers are single-stranded DNA or RNA oligonucleotidesequences with the capacity to recognize various target molecules withhigh affinity and specificity. These ligand-binding oligonucleotidesmimic properties of antibodies in a variety of diagnostic formats. Theyare folded into unique overall shapes to form intricate binding furrowsfor the target structure. Aptamers are identified by an in vitroselection process known as systematic evolution of ligands byexponential enrichment (SELEX). Aptamers may have advantages overantibodies in the ease of depositing them on sensing surfaces. Moreover,due to the highly reproducible synthetic approach in any quantities,albeit the affinity constants are consistently lower than those ofantibodies and the stability of these compounds is still questionable,they may be particularly useful for screening applications in complexbiological matrices (i.e., in identifying ACTH analogs with substitutedamino acid residues at ACTH-(15–19) and/or ACTH-(21) positions that areparticularly active in binding the MC2R).

Alternatively, molecular imprinting techniques may be used to screencompounds or methods that affect MC2R activity. This is a technique thatis based on the preparation of polymeric sorbents that are selectivelypredetermined for a particular substance, or group of structuralanalogs. Functional and cross-linking monomers of plastic materials,such as methacrylics and styrenes, are allowed to interact with atemplating ligand to create low-energy interactions. Subsequently,polymerization is induced. During this process, the molecule of interestis entrapped within the polymer either by a noncovalent, self-assemblingapproach, or by a reversible, covalent approach. After stopping thepolymerization, the template molecule is washed out. The resultantimprint of the template is maintained in the rigid polymer and possessesa steric (size, shape) and chemical (special arrangement ofcomplementary functionality) memory for the template. The molecularlyimprinted polymer (MIP) can bind the template (=analyte) with aspecificity similar to that of the antigen-antibody interaction.

Besides the main applications in solid-phase extraction andchromatography, molecularly imprinted polymers can be employed asnonbiological alternatives to antibodies in competitive binding assays.During the growth of a typical biological cell, carbon-containingnutrients such as glucose are taken up and acidic metabolic productssuch as lactic acid are released. In microphysiometry, these changes inmetabolic rate are recorded as changes in the rate of acidification ofthe medium surrounding the cells (i.e., Raley-Susman, K. M. et al.,“Effects of excitotoxin exposure on metabolic rate of primaryhippocampal cultures: application of silicon microphysiometry toneurobiology,” J Neurosci., 12(3):773–780 (1992); Baxter, G. T. et al.,“PKCε is involved in granulocyte-macrophage colony-stimulating factorsignal transduction: evidence from microphysiometry and antisenseoligonucleotide experiments,” Biochemistry, 31:19050–10954 (1992);Bouvier, C. et al., “Dopaminergic activity measured in D₁- and D₂-transfected fibroblasts by silicon microphysiometry,” J. Recept. Res.,13(1–4):559–571 (1993); and McConnell, H. M. et al., “The cytosensormicrophysiometer: biological applications of silicon technology,”Science, 257:1906–1912 (1992)). Microphysiometry can be used to detectmolecules that affect the cell. Such molecules includeneurotransmitters, growth factors, cytokins, receptors, and the like.Thus, the microphysiometry method can provide valuable informationregarding ACTH analogs that affect MC2R receptor activity.

Synthetic combinatorial libraries can be a source of diverse structuresuseful for large-scale biochemical screening (i.e., Sastry, L. et al.,“Screening combinatorial antibody libraries for catalytic acyl transferreactions,” Ciba Found Symp., 159:145–155 (1991); Persson, M. A. A. etal., “Generation of diverse high-affinity monoclonal antibodies byrepertoire cloning,” Proc. Natl. Acad. Sci. USA, 33:2432–2436 (1991);and Houghten, R. A., “Generation and use of synthetic peptidecombinatorial libraries for basic research and drug discovery,” Nature,354:84–86 (1991)). Such libraries are generated by a combination ofsolution and solid-phase chemistries and are cleaved off thesolid-support for screening.

Separation techniques such as liquid chromatography, gas chromatography,and capillary electrophoresis coupled to mass spectrometry ortandem-mass spectrometry create analytical systems available forstructural evaluation (i.e., Hsieh, S. et al., “Separation andidentification of peptides in single neurons by microcolumn liquidchromatography—Matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry and postsource decay analysis,” AnalChem., 70(9):1847–1852 (1998); Tretyakova, N. Y. et al., “Quantitativeanalysis of 1,3-butadene-induced DNA adducts in vivo and in vitro usingliquid chromatography electrospray ionization tandem mass spectrometry,”J. Mass Spectrom., 33:363–376 (1998); and Taylor, G. W. et al.,“Excursions in biomedical mass spectrometry,” Br. J. Clin. Pharmacol.,41:119–126 (1996)). Mass spectrometry is particularly useful inproviding information about the molecular weight of a compound/molecule.With refined and controlled fragmentation of large molecules, it is alsopossible to extract information about the sequence.

Methods of screening ACTH analog compounds to identify ACTH analogcompounds that induce less corticosteroid secretion by adrenal membranethan unmodified ACTH of SEQ ID NO:2 or SEQ ID NO:1 can include the stepof contacting an adrenal membrane with an ACTH analog. Methods ofscreening ACTH analog compounds to identify compounds with reducedACTH-induced secretion of corticosteroid can include one or more of thefollowing steps:

-   -   a. providing a first adrenal membrane and a second adrenal        membrane;    -   b. contacting the first adrenal membrane with a first        composition comprising an unmodified peptide comprising an        unmodified ACTH peptide, and subsequently measuring a first        concentration of corticosteroid secreted by the first adrenal        membrane after contact with the unmodified ACTH peptide;    -   c. contacting the second adrenal membrane with a second        composition comprising the ACTH analog, and subsequently        measuring a second concentration of corticosteroid secreted by        the second adrenal membrane after contacting the ACTH analog;    -   d. comparing the first concentration of corticosteroid secreted        with the second concentration of corticosteroid secreted; and    -   e. determining whether the second compound induces less        corticosteroid secretion than the first compound.        The unmodified ACTH peptide is preferably the peptide of SEQ ID        NO:2 or SEQ ID NO:1.

The first adrenal membrane and the second adrenal membrane can bepositioned within a subject and the concentration of corticosteroid canbe measured in the blood of the subject (in vivo assays). Alternatively,the first adrenal membrane and the second adrenal membrane are explantedfrom the subject and wherein the concentration of corticosteroid ismeasured in serum-free media (in vitro assays). Screening Assayscomprising in vivo Inhibition Assay steps or in vitro competitivebinding assay steps, can also include the following step, which ispreferably performed either in place of or after step (c) and beforestep (d):

-   -   f. simultaneously contacting the second adrenal membrane with        the first composition comprising the ACTH peptide and the second        composition comprising the ACTH analog, and subsequently        measuring a second concentration of corticosteroid secreted by        the second adrenal membrane.

Screening assays can include steps of performing one or more otherassays, including the assays described as the Serum CorticosteroidInduction Assay, Serum Corticosteroid Inhibition Assay, Adrenal BindingAssay, or Serum-Free Adrenal Inhibition Assay. Steps can be performed inany order, unless otherwise specified.

ACTH Analog Compositions and Administration

The ACTH analogs can be incorporated in pharmaceutical compositions fortreating various ACTH-related conditions, including conditions relatedto the over-expression of ACTH in patients.

The phrase “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer synonymously to ligands, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

A “therapeutically effective amount” of a compound such as an ACTHanalog peptide, with respect to use in treatment, refers to an amount ofthe polypeptide or peptide in a preparation which, when administered aspart of a desired dosage regimen (to a chordate, such as a mammal orfish) alleviates a symptom, ameliorates a condition, or slows the onsetof disease conditions according to clinically acceptable standards forthe disorder or condition to be treated or the cosmetic purpose, e.g.,at a reasonable benefit/risk ratio applicable to any medical treatment.

The term “treat” or any derivative thereof (i.e., treatment, treating),as used herein, refers to and includes: 1) preventing a disease orcondition associated with high levels of ACTH from occurring in apatient that may be predisposed to such disease or condition but has notyet been diagnosed as having it; 2) inhibiting the disease or conditionassociated with high levels of ACTH, e.g., arresting its development; or3) relieving the disease or condition associated with high levels ofACTH, e.g., causing regression of the disease or condition.

A “patient,” or “subject,” are used synonymously herein to refer to anorganism, preferably a chordate animal such as a mammal, fish or avianspecies, for which treatment is provided in accordance with the presentinvention. Mammalian species that benefit from the disclosed ACTHanalogs and methods of treatment include, but are not limited to, apes,chimpanzees, orangutans, humans, monkeys; and domesticated animals(e.g., pets) such as dogs, cats, mice, rats, guinea pigs, and hamsters.Fish species include salmon and other species used in aquaculture.

The term “ACTH-related condition” is used herein to refer to conditions,disorders, symptoms or diseases that may be treated by, or areresponsive to, the regulation of ACTH-mediated corticosteroid orcorticosteroid levels, regulation of ACTH-binding to adrenal membrane,or regulation of an ACTH-receptor such as MC-2R, including certainmelanocortin-receptor associated conditions.

The term “melanocortin-receptor associated condition” is used herein torefer to conditions, disorders, or diseases that may be treated byregulation of a receptor that binds ACTH, and/or by reduction incorticosteroid levels in a subject. An ACTH-related condition caninclude “MC-2R associated conditions,” or conditions, disorders ordiseases that may be treated by regulation of the MC-2R receptor.Preferably, MC-2R associated conditions can be treated by binding theMC-2R receptor with an ACTH analog that results in a reduced level ofcorticosteroid secretion compared to the level of corticosteroidsecretion by endogenous ACTH.

All stereoisomers of the compounds identified using methods of theinvention, including enantiomeric and diastereomeric forms, arecontemplated within the scope of this invention. Individualstereoisomers of the compounds of the invention may, for example, besubstantially free of other isomers, or may be admixed, for example, asracemates or with all other, or other selected, stereoisomers. Thechiral centers of the compounds identified by the present invention canhave the S or R, or D or L, configuration as defined by the IUPAC 1974Recommendations.

In an eighth embodiment, the present disclosure pertains topharmaceutical compositions comprising ACTH analogs, and theadministration thereof to a subject in a manner commensurate withtreatment for symptoms associated with an ACTH-related condition. Theroute of administration can be selected in accord with known methods.ACTH analogs can be employed as part of a pharmaceutical compositionincluding a pharmaceutically-acceptable carrier. The pharmaceuticalcompositions comprising at least one ACTH analog of the invention totreat a disease or condition associated with elevated levels of ACTH,may be formulated, for example, by employing conventional solid orliquid vehicles or diluents, as well as pharmaceutical additives of atype appropriate to the mode of desired administration (for example,excipients, binders, preservatives, stabilizers, flavors, etc.)according to techniques such as those well known in the art ofpharmaceutical formulation.

Pharmaceutical compositions comprising ACTH analogs can be administeredby any suitable means. For example, compositions comprising ACTH analogscan be administered by injection by subcutaneous, intravenous,intramuscular, or intracisternal injection or infusion techniques (e.g.,as sterile injectable aqueous or non-aqueous solutions or suspensions);nasally, such as by inhalation spray; or topically, such as in the formof a cream or ointment; and in dosage unit formulations containingnon-toxic, pharmaceutically-acceptable vehicles or diluents. ACTHanalogs of the invention may, for example, be administered in a formsuitable for immediate release or extended release. Immediate release orextended release may be achieved by the use of suitable pharmaceuticalcompositions comprising the ACTH analogs of the invention, or,particularly in the case of extended release, by the use of devices suchas subcutaneous implants or osmotic pumps. The ACTH analogs of theinvention may also be administered in the form of liposomes.

Pharmaceutical compositions comprising the ACTH analog polypeptides ofthe present invention can be formulated according to known methods,whereby the ACTH analog product hereof is combined in admixture with apharmaceutically acceptable carrier vehicle. Compositions which may beused for the prophylactic and therapeutic treatment include one or moreACTH analog compounds and a means of application (such as an injectablecarrier system, intranasal or transdermal mode).

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or subjectbeing exposed thereto at the dosages and concentrations employed. Thephrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ or portion of the body, to another organ or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, not injurious to thepatient, and substantially non-pyrogenic. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include: (1)sugars, such as lactose, glucose, and sucrose; (2) starches, such ascorn starch and potato starch; (3) cellulose, and its derivatives, suchas sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; (4) powdered kagacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;and (21) other non-toxic compatible substances employed inpharmaceutical formulations. In certain embodiments, pharmaceuticalcompositions of the present invention are non-pyrogenic, i.e., do notinduce significant temperature elevations when administered to apatient. Often, the physiologically acceptable carrier is an aqueous pHbuffered solution. Acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids or amino acid derivatives such asnorleucine or ornithene; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG. Any of the carriers for the ACTH analog may bemanufactured by conventional means. However, if alcohol is used in thecarrier, the ACTH analog should be in a micelle, liposome, or a“reverse” liposome, to prevent denaturing of the ACTH analog. Similarly,when the ACTH analog is being placed in the carrier, and the carrier is,or has been heated, such placement should be made after the carrier hascooled somewhat, to avoid heat denaturation of the ACTH analog. Thecarrier preferably is sterile. One or more ACTH analogs may be added tothese substances in a liquid form or in a lyophilized state, whereuponit will be solubilized when it meets a liquid body.

Prior to, or at the time the modified ACTH analog can be combined with acarrier system, the ACTH analog may be in a stabilizing bufferenvironment for maintaining a pharmacologically suitable pH range, suchas between about 4.0 and about 9.0, including a pH of about 5.0, 6.0,7.0, 8.0 or any pH interval of 0.05 there between, or any interval thatis a multiple of 0.05 there between, including pH values of 5.2, 6.5,7.4, 7.5 and 8.5. Therapeutic formulations are prepared for storage bymixing the ACTH analog active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. A stabilizing buffer should allow for the optimum activity ofthe ACTH analog. The buffer may contain a reducing reagent, such asdithiothreitol. The stabilizing buffer also may be or include a metalchelating reagent, such as ethylenediaminetetracetic acid disodium salt,or it also may contain a phosphate or citrate phosphate buffer, or anyother buffer.

The effective amount of a compound employed in the present invention maybe determined by one of ordinary skill in the art. The effective dosagerates or amounts of the ACTH analog(s) will depend in part on whetherthe ACTH analog(s) will be used therapeutically or prophylactically, theduration of exposure of the recipient to the infectious bacteria, thesize and weight of the individual, and other considerations within thescope of medical judgment. The duration for use of the compositioncontaining the ACTH analog also depends on whether the use is forprophylactic purposes, wherein the use may be hourly, daily or weekly,for a short time period, or whether the use will be for therapeuticpurposes wherein a more intensive regimen of the use of the compositionmay be needed, such that usage may last for hours, days or weeks, and/oron a daily basis, or at timed intervals during the day. Any dosage formemployed should provide for a minimum number of units for a minimumamount of time. Dosages and desired drug concentrations ofpharmaceutical compositions of the present invention may vary dependingon the particular use envisioned. For example, where an ACTH analog ofthe invention is to be administered to a patient to treat a Cushing'sSyndrome or premature labor, exemplary dosage amounts for an adult humaninclude from about 0.001 to 100 mg/kg of body weight of active compoundper day, which may be administered in a single dose or in the form ofindividual divided doses, such as from 1 to 4 times per day. It will beunderstood that the specific dose level and frequency of dosage for anyparticular compound may be varied and will depend upon a variety offactors including the activity of the specific compound employed, themetabolic stability and length of action of that compound, the species,age, body weight, general health, sex and diet of the patient, the modeand time of administration, rate of excretion, drug combination, andseverity of the particular condition.

The determination of the appropriate dosage or route of administrationis well within the skill of an ordinary physician. Animal-experimentsprovide reliable guidance for the determination of effective doses forhuman therapy. Interspecies scaling of effective doses can be performedfollowing the principles laid down by Mordenti, J. and Chappell, W. “Theuse of interspecies scaling in toxicokinetics” In Toxicokinetics and NewDrug Development, Yacobi et al., Eds., Pergamon Press, New York 1989,pp. 42–96. The concentration of the active units of ACTH analog that mayprovide for an effective amount or dosage of ACTH analog may be in therange of about 10 units/ml to about 500,000 units/ml of fluid in the wetor damp environment of the nasal and oral passages, and topically aswell and possibly in the range of about 10, 20, 30, 40, 50, 60, 70, 80,90, or 100 units/ml to about 50,000 units/ml. Representative values thusinclude about 200 units/ml, 300 units/ml, 500 units/ml, 1,000 units/ml,2,500 units/ml, 5,000 units/ml, 10,000 units/ml, 20,000 units/ml, 30,000units/ml, and 40,000 units/ml. More specifically, time exposure to theactive ACTH analog units may influence the desired concentration ofactive ACTH analog units per ml. The formulations to be used for in vivoadministration are preferably sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. Therapeutic compositions hereingenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Examples of pharmaceutical compositions comprising ACTH analogs includeinjectable compositions, compositions providing for the sustainedrelease of the ACTH analog over a desired time period, compositions fordermal administration, an injectable gel, coatings for an implantablemedical devices, mucoadhesive compositions, or compositions for nasal orother inhalable compositions.

In some examples, an ACTH analog may be administered by intramuscular orintravenous injection. For example, the ACTH analogs can be administeredintramuscularly, intravenously, subcutaneously, subdermally,intradermally or combinations thereof.

The injectable composition preferably comprises a suitable carrier andone or more ACTH analog compounds. The carrier may be comprised ofdistilled water, a saline solution, albumin, a serum, or anycombinations thereof. In cases where intramuscular injection is thechosen mode of administration, an isotonic formulation may be used.Generally, additives for isotonicity can include sodium chloride,dextrose, mannitol, sorbitol and lactose. In some cases, isotonicsolutions such as phosphate buffered saline are used. Stabilizersinclude gelatin and albumin. In some examples, a vasoconstriction agentis added to the formulation. The pharmaceutical preparations areprovided sterile and pyrogen free. Generally, as noted above,intravenous injection may be most appropriate. Exemplary compositionsfor parenteral administration include injectable solutions orsuspensions which may contain, for example, suitable non-toxic,parenterally acceptable diluents or solvents, such as mannitol,1,3-butanediol, water, Ringer's solution, an isotonic sodium chloridesolution, or other suitable dispersing or wetting and suspending agents,including synthetic mono- or diglycerides, and fatty acids, includingoleic acid. Glycerin or glycerol (1,2,3 propanetriol) is a commerciallyavailable carrier for pharmaceutical use. Glycerin or glycerol may bediluted in sterile water for injection, or sodium chloride injection, orother pharmaceutically acceptable aqueous injection fluid, and used inconcentrations of 0.1 to 100% (v/v), 1.0 to 50% or about 20%. DMSO, isan aprotic solvent that can enhance penetration of many locally applieddrugs. DMSO may be diluted in sterile water for injection, or sodiumchloride injection, or other pharmaceutically acceptable aqueousinjection fluid, and used in concentrations of 0.1 to 100% (v/v). Thevehicle also may include Ringer's solution, a buffered solution, anddextrose solution, particularly when an intravenous solution isprepared.

Prior to, or at the time the ACTH analog is put in the carrier system,it may be desirable for the ACTH analogs be in a stabilizing bufferenvironment, maintaining a suitable pH range. The stabilizing buffershould allow for the optimum activity of the ACTH analog. The buffer maybe a reducing reagent, such as dithiothreitol. The stabilizing bufferalso may be or include a metal chelating reagent, such asethylenediaminetetracetic acid disodium salt, or it also may contain aphosphate or citrate phosphate buffer. The buffers found in the carriercan serve to stabilize the environment for the ACTH analogs.

The carrier can optionally contain minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter ions such as sodium; nonionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or pharmaceutically acceptable salts, e.g., NaCl, KCl,MgCl₂, CaCl₂, and the like.

The term “pharmaceutically acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the—14 hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts, and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1–19.) In other cases, the inhibitors useful in the methods ofthe present invention may contain one or more acidic functional groupsand, thus, are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base; such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Injectable pharmaceutical formulations comprising ACTH analogs canoptionally include other therapeutic agents including antimicrobialagents, anti-inflammatory agents, antiviral agents, or local anestheticagents. Local anesthetics include tetracaine, tetracaine hydrochloride,lidocaine, lidocaine hydrochloride, dyclonine, dyclonine hydrochloride,dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride,butambenpicrate, and pramoxine hydrochloride. An exemplary concentrationfor local anesthetics is about 0.025% to about 5% by weight of the totalcomposition. Anesthetics such as benzocaine also may be used at apreferred concentration of about 2% to about 25% by weight.

Pharmaceutical compositions can also optionally comprise one or moreadditional bioactive agents to suppress the synthesis of steroidhormones at various levels (i.e. inhibitors of enzymes which catalyzevarious stages of the synthesis of steroid hormones), including thebioactive agents reviewed in J.Steroid Biochem., vol. 5, p. 501 (1974)which include the following: a) derivatives of diphenylmethane, e.g.amphenon B (which suppresses the synthesis of steroid hormones at stages11-beta-, 17- and 21- of hydroxylase); b) derivatives of pyridine (SU-cseries), e.g. metirapon (which suppresses synthesis at stage 11-beta ofhydroxylase); c) substituted alpha, alpha-glutaramides, e.g.aminoglutetimide (which impedes the synthesis of pregnenolone fromcholesterol through suppression of 20-alpha-hydroxylase and C20,C22-liase; d) steroid substances e.g. trilostan (3 beta-substitutedsteroid-3beta-hydroxy-5-androsten-17-one), which suppresses 3beta-desoxysteroidhydrogenase-5.4-isomerase (Steroids, vol. 32, p. 257);e) steroids of the spironolactone family which are used as rapidlydissociating anti-Mineralocorticoids (PNAS USA 71(4) p. 1431–1435(1974)); f) synthetic steroid described as an anti-Mineralocorticoids,ZK91587, showing specific binding properties for the kidney(Z.Naturforsch., 45b, p. 711–715 (1990)) and hippocampus type I MR (LifeScience, 59, p. 511–21 (1996)), but not for type II GR. It may thereforebe conveniently useful as a tool in the investigation of MR function intissues containing both receptor systems.

ACTH analogs can be optionally administered with bioactive agents thatspecifically suppress the interaction of glucocorticoid hormones withhormone receptors include: a) Mifepriston (11β,17β)-11-[4-(Dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one,which acts on receptors of glucocorticoid hormones to form a complexincapable of initiating mechanisms leading to glucocorticoid effect(Annals of New-York Academy of Science, vol. 761, p. 296–310 (1995));said compound is also known as a contragestive agent (RU38486 or RU486);b) non-steroid substances (J:Steroid Biochem., vol. 31, p. 481–492(1988)) e.g. drotaverina hydrochloride (a derivative ofisoquinoline-1-(3.4-dietoxibenezilidene)-6.7-dietoxy-1,2,3,4-tetrahydrizoquinoline) or acetvisalicicacid (Moskovskava Meditsina, 1990, “Receptor mechanisms of theglucocorticoid effect” by V. P. Golikov). Antiglucocorticoids (e.g.Mifepristone) have been used in a clinical setting is to treatinoperable cases of nonpituitary Cushing's syndrome. In the case ofMifepristone (both an anti-progesterone and an anti-glucocorticoid),high doses (up to 800 mg per day) can be required. Employing asystematic application of strategies to increase activity and decreasecross-reactivity and undesirable side effects, impressive progress hasbeen reported in the development of new antihormonal agents with greaterpotency and selectivity, especially in the antiestrogen and antiandrogenfields.

The effective dosage rates or amounts of the ACTH analog to beadministered parenterally, and the duration of treatment will depend inpart on the seriousness of the infection, the weight of the patient, theduration of exposure of the recipient to the infectious bacteria, theseriousness of the infection, and a variety of a number of othervariables. The composition may be applied anywhere from once to severaltimes a day, and may be applied for a short or long term period. Theusage may last for days or weeks. Any dosage form employed shouldprovide for a minimum number of units for a minimum amount of time. Theconcentration of the active units of ACTH analog believed to provide foran effective amount or dosage of ACTH analog may be in the range ofabout 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units/ml to about10,000,000 units/ml of composition, in a range of about 1000 units/ml toabout 10,000,000 units/ml, and from about 10,000 to 10,000,000 units/ml.The amount of active units per ml and the duration of time of exposuredepend on the nature of infection, and the amount of contact the carrierallows the ACTH analog to have. It is to be remembered that the ACTHanalog works best when in a fluid environment. Hence, effectiveness ofthe ACTH analog is in part related to the amount of moisture trapped bythe carrier. The concentration of the ACTH analog for the treatment isdependent upon the bacterial count in the blood and the blood volume.

When in vivo administration of an ACTH analog is employed, normal dosageamounts may vary from about 10 ng/kg to up to 100 mg/kg of subject bodyweight or more per day, or about 1 pg/kg/day to 10 mg/kg/day, dependingupon the route of administration. Guidance as to particular dosages andmethods of delivery is also provided below, as well as in theliterature. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue.

Pharmaceutical solutions for infusion or injection may be prepared in aconventional manner, e.g. with the addition of preservatives such asp-hydroxybenzoates or stabilizers such as alkali metal salts of ethylenediamine tetraacetic acid, which may then be transferred into fusionvessels, injection vials or ampules. Alternatively, the compound forinjection may be lyophilized either with or without the otheringredients and be solubilized in a buffered solution or distilledwater, as appropriate, at the time of use. Non aqueous vehicles such asfixed oils, liposomes, and ethyl oleate are also useful herein.

Additionally, a number of methods can be used to assist in transportingthe ACTH analog across the cell membrane. The ACTH analog can betransported in a liposome, with the ACTH analog “inserted” in theliposomes by known techniques. Similarly, the ACTH analog may be in areverse micelle. The ACTH analog can also be pegylated, attaching thepolyethylene glycol to the non-active part of the ACTH analog.Alternatively, hydrophobic molecules can be used to transport the ACTHanalog across the cell membrane. Finally, the glycosylation of the ACTHanalog can be used to target specific internalization receptors on themembrane of the cell.

Materials having controlled release capability can be particularlydesirable for certain methods of treatment. The ACTH analog can becombined with a carrier system such as a polymer that provides forsustained release of the ACTH analog compound over a desired period oftime can be used. Also included in such formulations may be highmolecular weight excipients such as celluloses (avicel) or polyethyleneglycols (PEG). Such formulations may also include an excipient to aidmucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy propylmethyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleicanhydride copolymer (e.g., Gantrez), and agents to control release suchas polyacrylic copolymer (e.g., Carbopol 934). Lubricants, glidants,flavors, coloring agents and stabilizers may also be added for ease offabrication and use.

ACTH analog compounds can be combined with the sustained release carriercomposition described by Loughman in U.S. Pat. No. 6,893,645, filed Jul.15, 2002 and issued May 17, 2005, which is incorporated herein byreference. Other examples of sustained or controlled releaseformulations and methods that can be combined with ACTH analogs includePublished U.S. Patent Application Nos. US2005/0164927A1, filed Aug. 18,2004, by Cheung et al., US2003/0125528A1 filed Sep. 27, 2002 by Hay etal., US2003/0211966A1, filed Oct. 21, 2002 by Kubek et al. andUS2003/0148938A1, filed Nov. 7, 2001 by Sharma et al., andUS2003/0181361A1, filed Mar. 31, 2003 by Sharma et al., the disclosuresof which are incorporated herein by reference in their entirety. In someembodiments, a controlled release treatment composition can include acontrolled release polymer and an effective amount of an ACTH analog.The controlled release polymer can be a water swellable polymer,optionally including a desired level of cross linking moieties. Thecontrolled release polymer can have a desired level of water solubility,or have minimal water solubility.

Polymer thickeners that may be used include those known to one skilledin the art, such as hydrophilic and hydroalcoholic gelling agentsfrequently used in the cosmetic and pharmaceutical industries. Thehydrophilic or hydroalcoholic gelling agent can comprise, for example,“CARBOPOL®” (B.F. Goodrich, Cleveland, Ohio), “HYPAN®” (KingstonTechnologies, Dayton, N.J.), “NATROSOL®” (Aqualon, Wilmington, Del.),“KLUCEL®” (Aqualon, Wilmington, Del.), or “STABILEZE®” (ISPTechnologies, Wayne, N.J.). The gelling agent may comprise between about0.2% to about 4% by weight of the composition. More particularly, anexample of the compositional weight percent range for “CARBOPOL®” may bebetween about 0.5% to about 2%, while the weight percent range for“NATROSOL®” and “KLUCEL®” may be between about 0.5% to about 4%. Acompositional weight percent range for both “HYPAN®” and “STABILEZE®”may be between about 0.5% to about 4%.

“CARBOPOL®” is one of numerous cross-linked acrylic acid polymers thatare given the general adopted name carbomer. These polymers dissolve inwater and form a clear or slightly hazy gel upon neutralization with acaustic material such as sodium hydroxide, potassium hydroxide,triethanolamine, or other amine bases. “KLUCEL®” is a cellulose polymerthat is dispersed in water and forms a uniform gel upon completehydration. Other gelling polymers include hydroxyethylcellulose,cellulose gum, MVE/MA decadiene crosspolymer, PVM/MA copolymer, or acombination thereof.

Preservatives also may be used in this invention and may comprise, forexample, about 0.05% to 0.5% by weight of the total composition. The useof preservatives assures that if the product is microbiallycontaminated, the formulation will prevent or diminish microorganismgrowth. Some preservatives useful in this invention includemethylparaben, propylparaben, butylparaben, chloroxylenol, sodiumbenzoate, DMDM Hydantoin, 3-Iodo-2-Propylbutyl carbamate, potassiumsorbate, chlorhexidine digluconate, or a combination thereof.

Where sustained-release administration of an ACTH analog is desired in aformulation with release characteristics suitable for the treatment ofany disease or disorder requiring administration of the ACTH analog,microencapsulation of the ACTH analog is contemplated. Perferredmicroencapsulation compositions suitable for use with the ACTH analogsinclude those described in U.S. Pat. No. 6,475,507 (filed Nov. 6, 2000)to Pellet et al., and U.S. Pat. No. 6,911,218 (filed Apr. 20, 1999) toIgnatious et al., both of which are incorporated herein by reference intheir entirety. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon-(rhIFN—), interleukin-2, and MN rgp120. Johnson etal., Nat. Med., 2:795–799 (1996); Yasuda, Biomed. Ther., 27:1221–1223(1993); Hora et al., Bio/Technology. 8:755–758 (1990); Cleland, “Designand Production of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems.” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

Sustained or control release carriers can be “long” or “slow” releasecarriers (such as, for example, certain nasal sprays, polymers, orcapsules) could possess or provide a lower concentration of active (ACTHanalog) units per ml, but over a longer period of time, whereas a“short” or “fast” release carrier (such as, for example, a gargle) couldpossess or provide a high concentration of active (ACTH analog) unitsper ml, but over a shorter period of time. The amount of active unitsper ml and the duration of time of exposure depend on the nature ofinfection, whether treatment is to be prophylactic or therapeutic, andother variables. Thus, the number of dosages will be dependent upon thecircumstances and can range from 1–4 times per day or more, withdurations from one day to multiple weeks. Infections can occur in theskin and thus such compositions may be formulated for topicalapplication as well, using well known vehicles such as those describedin U.S. Pat. Nos. 6,056,954 and 6,056,955. Micelles and multi lamellarmicelles also may be used to control the release of ACTH analog.

The sustained-release formulations of these proteins can use abioabsorbable polymer, such as poly-lactic-coglycolic acid (PLGA)polymer due to its biocompatibility and wide range of biodegradableproperties. The degradation products of. PLGA, lactic and glycolicacids, can be cleared quickly within the human body. Moreover, thedegradability of this polymer can be adjusted from months to yearsdepending on its molecular weight and composition. Lewis, “Controlledrelease of bioactive agents from lactide/glycolide polymer,” in: M.Chasin and R. Langer (Eds.), Biodegradable Polymers as Drul: DeliverySystems (Marcel Dekker: New York, 1990), pp. 1–41.

In some examples, a therapeutic composition comprises a mucoadhesivesustained release formulation and an ACTH analog. The mucosal lining, asdisclosed and described, includes, for example, the upper and lowerrespiratory tract, eye, buccal cavity, nose, rectum, vagina, periodontalpocket, intestines and colon. Orahesive® from E.R. Squibb & Co is anadhesive which is a combination of pectin, gelatin, and sodiumcarboxymethyl cellulose in a tacky hydrocarbon polymer, for adhering tothe oral mucosa. However, such physical mixtures of hydrophilic andhydrophobic components eventually fall apart. In contrast, thehydrophilic and hydrophobic domains in the present disclosure produce aninsoluble copolymer. U.S. Pat. No. 4,948,580, also incorporated byreference, describes a bioadhesive oral drug delivery system. Thecomposition includes a freeze dried polymer mixture formed of thecopolymer poly(methyl vinyl ether/maleic anhydride) and gelatin,dispersed in an ointment base, such as mineral oil containing dispersedpolyethylene. U.S. Pat. No. 5,413,792 (incorporated herein by reference)discloses paste like preparations comprising (A) a paste like basecomprising a polyorganosiloxane and a water soluble polymeric materialwhich are may be present in a ratio by weight from 3:6 to 6:3, and (B)an active ingredient. U.S. Pat. No. 5,554,380 claims a solid orsemisolid bioadherent orally ingestible drug delivery system containinga water in oil system having at least two phases. One phase comprisesfrom about 25% to about 75% by volume of an internal hydrophilic phaseand the other phase comprises from about 23% to about 75% by volume ofan external hydrophobic phase, wherein the external hydrophobic phase iscomprised of three components: (a) an emulsifier, (b) a glyceride ester,and (c) a wax material. U.S. Pat. No. 5,942,243 describes somerepresentative release materials useful for administering antibacterialagents, which disclosure is incorporated by reference. The dosage formsof the compositions of this disclosure can be prepared by conventionalmethods.

Compositions comprising an ACTH analog may be administered by nasalsprays, nasal drops, nasal ointments, nasal washes, nasal injections,nasal packings, bronchial sprays and inhalers. When the ACTH analog(s)is introduced directly by use of nasal sprays, nasal drops, nasalointments, nasal washes, nasal injections, nasal packing, bronchialsprays, oral sprays, and inhalers, the ACTH analog may be in a liquid orgel environment, with the liquid acting as the carrier. A dry anhydrousversion of the modified ACTH analog may be administered by the inhalerand bronchial spray, although a liquid form of delivery also may beused. The nasal spray can be a long acting or timed release spray, andcan be manufactured by means well known in the art. An inhalant also maybe used, so that the ACTH analog may reach further down into thebronchial tract, including into the lungs. Exemplary compositions fornasal aerosol or inhalation administration include solutions in salinewhich may contain, for example, benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability, and/orother solubilizing or dispersing agents such as those known in the art.

Compositions for dermal administration of an ACTH analog can beformulated using a carrier for delivering at least one ACTH analog to orthrough the skin. The mode of application for the ACTH analog includes anumber of different types and combinations of carriers which include,but are not limited to an aqueous liquid, an alcohol base liquid, awater soluble gel, a lotion, an ointment, a nonaqueous liquid base, amineral oil base, a blend of mineral oil and petrolatum, lanolin,liposomes, protein carriers such as serum albumin or gelatin, powderedcellulose carmel, and combinations thereof. A mode of delivery of thecarrier containing the therapeutic agent includes, but is not limited toa smear, spray, a time-release pACTH, a liquid absorbed wipe, andcombinations thereof. The ACTH analog may be applied to a bandage eitherdirectly or in one of the other carriers. The bandages may be sold dampor dry, wherein the ACTH analog is in a lyophilized form on the bandage.This method of application is most effective for the treatment ofinfected skin. The carriers of topical compositions may comprisesemi-solid and gel-like vehicles that include a polymer thickener,water, preservatives, active surfactants or emulsifiers, antioxidants,sun screens, and a solvent or mixed solvent system. U.S. Pat. No.5,863,560 (Osborne) discusses a number of different carrier combinationsthat can aid in the exposure of the skin to a medicament. Anothercomposition for topical administration includes a topical carrier suchas Plastibase (mineral oil gelled with polyethylene).

In one example, the invention comprises a dermatological compositionhaving about 0.5% to 10% carbomer and about 0.5% to 10% of apharmaceutical that exists in both a dissolved state and a microparticulate state. The dissolved pharmaceutical has the capacity tocross the stratum corneum, whereas the micro particulate pharmaceuticaldoes not. Addition of an amine base, potassium, hydroxide solution, orsodium hydroxide solution completes the formation of the gel. Moreparticularly, the pharmaceutical may include dapsone, an antimicrobialagent having anti-inflammatory properties. One exemplary ratio of microparticulate to dissolved dapsone is five or less.

In another example, the invention comprises about 1% carbomer, about80–90% water, about 10% ethoxydiglycol, about 0.2% methylparaben, about0.3% to 3.0% dapsone including both micro particulate dapsone anddissolved dapsone, and about 2% caustic material. More particularly, thecarbomer may include “CARBOPOL® 980” and the caustic material mayinclude sodium hydroxide solution.

Methods of Treatment

The ACTH analog Compounds can be used to treat a variety of ACTH-relatedconditions. Methods of treatment comprising administration of one ormore ACTH analog compounds to reduce the rate of ACTH-induced adrenalhormone secretion, to mitigate the effects of high levels of ACTH inpatients or to block excess ACTH while maintaining a tonic state ofadrenal function. The ACTH analog compounds can be useful in treatingdiseases relating to levels of ACTH, such as conditions responsive tomodulation of ACTH receptors (such as MC-2R). The ACTH analog compoundscan be administered to treat conditions related to the regulation ofACTH levels, for example to decrease the effects of high levels of ACTHin patients while maintaining a tonic state of adrenal function.

ACTH analog peptides can be administered in methods of treating acondition presenting one or more symptoms of an ACTH-related conditionare provided. Symptoms of ACTH-related conditions include thosedescribed herein, as well as those known based on medical judgment andtherapeutic diagnostic practice, procedures and analysis. The methodspreferably include the step of administering to a subject apharmaceutical composition comprising an ACTH analog peptide. The ACTHanalog compositions are useful, for example, in treating ACTH-relatedconditions, such as Cushing's Syndrome, impaired immune response as aresult of hypersecretion of corticosteroid, initiation of prematurelabor (for example, by the hypothalmus-pituitary-adrenal axis), andrelated conditions. In one aspect, various ACTH analogs are prepared andadministered to a patient to assess in vivo cortisone induction. ACTHanalog compounds can also be administered to treat ACTH-producing tumorsin the pituitary gland, in combination with high resolution MR pituitaryimaging. ACTH analog compounds can also be administered in combinationwith petrosal sinus sampling to establish pituitary-derived ACTHhyper-secretion, pre-operative pituitary tumor localization andlateralization (see Oldfield, E. W. et al., “Petrosal sinus samplingwith and without corticotrophin-releasing hormone for the differentialdiagnosis of Cushing's syndrome,” N Engl J Med., 325:897–905 (1991); andFindling, J. W. et al., Endocrinol Metab Clin North Am., 30:729–47(2001)). Although 70% of pituitary microaderiomas are successfullyresected by tranfssphenoidal approaches, surgical “cure” rates formacroadenomas are achieved in only about a third of patients inspecialized centers (Mampalam, T. J. et al., Ann Intern Med., 109:487–93(1988)).

ACTH analog compounds can also be administered in tumor treatments,including in conjunction with treatment for ACTH hypersecretion, or incombination with pituitary-directed radiation to suppress tumor growthand hormonal levels. Radiation effects may not be manifest for severalyears, and are ultimately associated with pituitary damage anddysfunction in most patients (Brada, M. et al., “The long-term efficacyof conservative surgery and radiotherapy in the control of pituitaryadenomas,” Clin Endocrinol., 38:571–8 (1993)). Hypercortisolism,cortisol hypersecretion, may be completely resolved by adrenalectomy(surgical removal of one or both of the adrenal glands), but thisapproach does not suppress pituitary tumor growth, and is alsoassociated with other co-morbidity (see Trainer, P. J. et al.,“Cushing's syndrome: Therapy directed at the adrenal glands,” EndocrinolMetab Clin North Am., 23:571–584 (1994)).

ACTH analog compounds can be administered in combination with medicaltherapy, such as co-administration of cyproheptadine, an anti-serotoninagent, used to suppress ACTH secretion but ultimate efficacy was poor,and its use has been discontinued (Krieger, D. T. et al.,“Cyproheptadine-induced remission of Cushings disease,” N Engl J Med.293:893–6 (1975)). Although the antifungal, ketoconazole, suppressesadrenal cortisol biosynthesis, the drug does not inhibit pituitary tumorgrowth or ACTH secretion, and idiosyncratic hepatic impairment limitsits longterm use (see Sonino, N., “The use of ketoconazole as aninhibitor of steroid production,” N Engo J Med., 317:812–8 (1987)).

During pregnancy, CRH can be produced by the placenta and fetalmembranes in substantial amounts during the third trimester of pregnancy(see Frim, D. M. et al., “Characterization and gestational regulation ofcorticotrophin-releasing hormone messenger RNA in human placenta,” JClin Invest., 82:287–292 (1988)), giving rise to an increase in CRHconcentrations in maternal peripheral plasma, particularly after 30weeks gestation (see Sasaki, A. et al., “Immunoreactivecorticotropin-releasing hormone in human plasma during pregnancy, labor,and delivery,” J Clin Endocrinol Metab., 64:224–229 (1987); and Goland,R. S. et al., “High levels of corticotropin-releasing hormoneimmunoreactivity in maternal and fetal plasma during pregnancy,” J ClinEndocrinol Metab., 63:119–1204 (1986)). Recent studies have suggestedthat maternal plasma levels of CRH are elevated in women with pretermlabor (see Wolfe, C. D. A. et al., “Plasma corticotrophin releasingfactor (CRF) in abnormal pregnancy,” Br J Obstet Gynaecol., 95:1003–1006(1988); Warren, W. B. et al, “Elevated maternal plasmacorticotropin-releasing hormone levels in pregnancies complicated bypreterm labor,” Am J Obstet Gynecol., 166:1198–1207 (1992); and McLean,M. et al., “A placental clock controlling the length of humanpregnancy,” Nat Med., 1:460–463 (1995)) and lower in those destined togive birth postterm (McLean et al., supra.).

ACTH analogs may be used in combination with tocolytic, orlabor-preventing, drugs to postpone labor. Examples of tocolytic drugsinclude Ritodrine and Terbutaline, which are beta-sympathomimetic.However, in order to be effective, these drugs must generally beadministered before the full onset of labor, and are of uncertainefficacy or safety if administered after labor has begun. Althoughtocolytic drugs are sometimes continuously administered at low levels toa pregnant woman, normally by infusion, during the duration of a highrisk period of her pregnancy, normally between about twenty-eight andthirty-four weeks gestation (28–34 weeks), the drugs can haveundesirable side effects upon renal function, respiratory function,heart rate and general body musculature tone. Dosages often must remainlow, such as up to about 0.1 cc per hour of 1 mg/cc solutionTerbutaline. The effectiveness of these low dosages in avoiding theonset of labor is inconsistent, and poorly quantified. Accordingly,there is a need for new treatments and methods for treating prematurelabor. More importantly, new treatments and methods for premature laborare needed that do not present undesirable side-effects.

In one embodiment, ACTH analog compositions can be administered toreduce biosynthesis of ACTH. This method of treatment can be used incombination with, or preferably instead of, the administration ofpharmaceutical agents such as Metryapone. Preferably, ACTH analogcompositions can be administered to treat incidence of Cushing's diseasethat develop during pregnancy, for example as described in: J. R.Lindsay and L. K. Nieman (2005) “The Hypothalamic-Pituitary-Adrenal Axisin Pregnancy: Challenges in Disease Detection and Treatment,” EndocrineReviews 26: 775–800). Treatment for this condition, usually to prolongpregnancy or to prepare for delivery, can include administration of anACTH polypeptide and/or Metryapone, which has been observed to be welltolerated without adverse effects on maternal hepatic function or fetaldevelopment. The patents can be administered metryapone to slow down thebiosynthesis of ACTH. ACTH analog compositions are particularlypreferred when the cause of the hypersecretion of corticosteroid isbelieved to be a result of the elevated levels of ACTH (presumably froma pituitary tumor in these women), for example as part of a strategy todecrease the synthesis of corticosteroid.

In another aspect, methods for treating veterinary subjects areprovided, such as methods for decreasing stress hormones to benefit thehealth of agricultural and aquacultural species grown at high populationdensities. Compositions comprising one or more ACTH analog compounds canbe administered in a method for decreasing stress hormones to benefitthe health of agricultural and aquacultural species grown at highpopulation densities.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the materials and techniques disclosed in the examples which followrepresent materials techniques discovered by the inventors to functionwell in the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

Example 1 Serum Corticosteroid Induction Assay (in vivo)

ACTH analogs with reduced ACTH-mediated secretion of bloodcorticosteroid can be identified by performing an in vivo SerumCorticosteroid Induction Assay to measure the blood corticosteroid levelin a subject after administration of the ACTH analog.

The in vivo Serum Corticosteroid Induction Assay was performed accordingthe following method. First, two to three month-old male FVB/N mice fromour colony, five per group, were injected intraperitoneally withdexamethasone (0.4 mg/0.1 ml PBS per mouse; Sigma, St. Louis, Mo.) tosuppress their endogenous ACTH production. See Hajos, G T et al.,“Studies of the potency of polypeptides with ACTH action by a new methodbased on continuous measurement of plasma corticosterone,” SteroidsLipids Res 3:225–228 (1972), Costa, J L, et al., “Mutational analysis ofevolutionarily conserved ACTH residues,” Gen Comp Endocrinol, 136:12–16(2004), and Karpac, J, et al., “Development, Maintenance, and Functionof the Adrenal Gland in Early Postnatal Pro-opiomelanocortin Null MutantMice,” Endocrinology (2005), published online ahead of print Feb. 24,2005, all of which are incorporated herein by reference.

Second, ninety to one hundred twenty minutes later, thedexamethasone-suppressed mice were injected subcutaneously between theshoulder blades with either unmodified mACTH(1–24) or one of severalACTH analog peptide compounds (1 μg/0.1 mL PBS/0.5% BSA), or withvehicle alone (control) (0.1 mL PBS/0.5% BSA). The following ACTH analogpeptide compounds were administered to separate mice: 1: murineACTH1–24; 2: V26F,E30K; 3: P19W,K21E,Y23R; 4: E30K,P36R; 5: ALA19–24; 6:V26F,P36R; 7: P19W, K21E; 8: P19W,K21A,delY23; 9: P19W,K21A; 10:KRRP16-19RMW (AT814) (SEQ ID NO:20).

Third, one hour later, blood was collected within less than 1 minutefrom a small incision in the tail, and serum was flash-frozen and storedat −80° C. until assayed. Serum corticosterone was determined bycompetitive radioimmunoassay (¹²⁵I RIA kit; ICN, Costa Mesa, Calif.)according to the manufacturer's recommendations. One microliter of serumwas used per assay sample. Samples were run in duplicates. The resultsfor the ACTH analog peptides described above are presented in the graphof FIG. 1, showing activity as a percentage of ACTH activity. These ACTHanalogs showed less activity than native ACTH.

Example 2 Serum Corticosteroid Inhibition Assay (in vivo)

ACTH analogs with reduced ACTH-mediated secretion of bloodcorticosteroid can be identified by performing an in vivo SerumCorticosteroid Inhibition Assay to test their ability to inhibit adrenalhormone production induced by unmodified ACTH. The blood corticosteroidlevel in a subject was measured after administration of an ACTH analogtest compound in combination with a compound with knowncorticosteroid-induction activity, such as ACTH or another ACTH analog.The ACTH analog test compound can be administered before, concurrentlywith, or after the corticosteroid-producing compound.

The in vivo Serum Corticosteroid Inhibition Assay was performedaccording the following method. The ACTH analog test compounds, such asthose showing low activities in Example 1, were tested for theirabilities to inhibit adrenal hormone production induced by unmodifiedACTH.

First, two to three month-old male FVB/N mice from our colony, five pergroup, were injected intraperitoneally with dexamethasone (0.4 mg/0.1 mlPBS per mouse; Sigma, St. Louis, Mo.) to suppress their endogenous ACTHproduction.

Second, ninety to one hundred twenty minutes later, animals wereinjected subcutaneously between the shoulder blades with wild type ACTHand/or test ACTH analog peptides (1 μg/0.1 mL PBS/0.5% BSA) or withvehicle alone (0.1 mL PBS/0.5% BSA).

Third, one hour later, blood was collected within less than 1 minutefrom a small incision in the tail, and serum was flash-frozen and storedat −80° C. until assayed. Serum corticosterone was determined bycompetitive radioimmunoassay (¹²⁵I RIA kit; ICN, Costa Mesa, Calif.)according to the manufacturer's recommendations. One microliter of serumwas used per assay sample. Samples were run in duplicates.

Results are shown graphically in FIG. 2, as described above, for theanalog AT814 (SEQ ID NO:20). Potency of peptides is expressed as percent induction of corticosterone, with native mouse ACTH being 100%.Comparison of corticosterone induction 1 hour after the last peptideinjection showed that AT814 reduces corticosterone induction to 35% ofthat of wildtype ACTH when applied 30 minutes before the ACTH injection,and to 10% of that of wildtype ACTH when applied at the same time as theACTH injection.

Example 3 Adrenal Binding Assays (in vitro)

ACTH analogs with the ability to bind adrenal receptors can beidentified by performing an in vitro Adrenal Binding Assay. The amountof a radiolabelled ACTH analog test compound binding to explantedadrenal membrane can be measured to identify ACTH analogs with adrenalreceptor binding activity. Preferably this is performed in a serum freemedium (in vitro Serum-free Adrenal Binding Assay)

ACTH analogs with the ability to bind adrenal receptors with a greateraffinity than compounds with known adrenal binding activity (preferablyACTH) can also be identified by performing an in vitro AdrenalCompetitive Binding Assay, whereby measurement of the reduction in theamount of the radiolabelled adrenal-binding compound (e.g., ACTH) ismeasured at various concentrations of non-radio-labeled ACTH analog testcompound in a medium containing explanted adrenal membrane. Thereduction in adrenal membrane binding by the radio-labelled adrenalbinding compound can be used to identify and characterize the bindingactivity of the ACTH analog test compound. In a series of in vitroAdrenal Competitive Binding assays, the reduction in binding byradio-labelled ACTH was measured in combination with addition of an ACTHanalog test compound to the adrenal membrane medium.

The in vitro Serum-free Adrenal Competitive Binding Assay was performedaccording the following method. First, adrenals were removed from mice,dissected free of fat, and cut in half. See Costa, J L, et al.,“Mutational analysis of evolutionarily conserved ACTH residues,” GenComp Endocrinol. 136:12–16 (2004); and Weber, M M, et al., “Postnataloverexpression of insulin-like growth factor II in transgenic mice isassociated with adrenocortical hyperplasia and enhancedsteroidogenesis,” Endocrinology 140:1537–1543 (1999), incorporatedherein by reference.

Second, each adrenal half was placed into an individual well of a 4-welldish, one dish per mouse. Adrenal halves were incubated at 5% CO2, 37°C., in 0.5 ml serum-free medium (M199; Invitrogen) for 30 minutes toequilibrate.

Third, the adrenal halves were macerated or otherwise dissociated usingDounce Homogenizer to form a membrane fraction. The fraction wasre-suspended in suitable buffer (Tris or HEPES). ¹²⁵I Radio-labelledACTH (200 ml) and non-radiolabelled test compound (e.g., ACTH, or anACTH analog such as AT814 (SEQ ID NO:20)) were then added to there-suspended membrane fraction in an amount to provide a totalconcentration of 0 nM, 10 nM, 100 nM and 1000 nM of the test compound;controls had no peptides added. The membrane fraction is recovered andradio signal was detected using a gamma detector. Reduction in theradio-signal detected after addition of the non-radiolabelled testcompounds can be correlated to binding affinity of the various testcompounds. A reduction in the radio-signal after addition of ACTH analogpeptide test compounds can indicate an increased affinity for binding toadrenal ACTH receptors such as MC-2R, and the ability to displace ACTHat the adrenal MC-2R receptor.

FIG. 3 displays the results of a series of in vitro Serum-free AdrenalCompetitive Binding Assays.

Example 4 Adrenal Inhibition Assay (in vitro)

ACTH analogs that reduce or block the ACTH-mediated secretion ofcorticosteroid from adrenal membrane can be identified by performing anin vitro Serum-free Corticosteroid Inhibition Assay to test theirability to inhibit adrenal hormone production induced by unmodifiedACTH. The corticosteroid level in a serum free media was measured afteradding an ACTH analog test compound in combination with a compound withknown corticosteroid-induction activity, such as ACTH or another ACTHanalog. The ACTH analog test compound can be added before, concurrentlywith, or after the corticosteroid-producing compound.

The in vitro Serum-free Adrenal Inhibition Assay was performed accordingthe following method. First, adrenals were removed from mice, dissectedfree of fat, and cut in half. See Costa, J L, et al., “Mutationalanalysis of evolutionarily conserved ACTH residues,” Gen CompEndocrinol. 136:12–16 (2004); and Weber, M M, et al., “Postnataloverexpression of insulin-like growth factor II in transgenic mice isassociated with adrenocortical hyperplasia and enhancedsteroidogenesis,” Endocrinology 140:1537–1543 (1999), incorporatedherein by reference.

Second, each adrenal half was placed into an individual well of a 4-welldish, one dish per mouse. Adrenal halves were incubated at 5% CO2, 37°C., in 0.5 ml serum-free medium (M199; Invitrogen) for 30 minutes toequilibrate.

Third, medium was removed and replaced by 0.5 ml M199 containing ACTH,AT814, or both, at 100 ng/ml each; controls had no peptides added.

Fourth, after 2 hours of incubation, the medium was removed, pooled forall four halves from each mouse, and assayed by corticosterone usingstandard RIA methods. corticosterone was determined by competitiveradioimmunoassay (¹²⁵I RIA kit; ICN, Costa Mesa, Calif.) according tothe manufacturer's recommendations. One microliter of serum was used perassay sample. Samples were run in duplicates. FIG. 4 displays theresults.

Example 5 (Predicted) Therapeutic Veterinary Administration

Fish hatcheries face a number of challenges as the goal of maximizingthe total tonnage of fish for commercial purposes is confounded by theeffects of crowding, and the spread of infection. In these environments,fish attempt to manage chronic stress insults through thehypothalamus/pituitary/interrenal (HPI) axis. In these situations a risein corticosteroid, is usually flowed by some level of mortality, adesensitization to corticosteroid and then a new steady-state withrespect to population density. Ironically during these “adjustment”cycles an extended rise in corticosteroid levels lowers resistance toinfection.

The HPI in hatchery fish can be modulated by the time releaseadministration of an ACTH analog of the invention, such as time releaseadministration from a silastic capsule implant in these fish. Thisbuffering of the HPI axis should improve survival and may facilitateweight gain in hACTHery fish.

Fish implanted with either the ACTH analog of the invention, or ACTHalone, would be subjected to environmental conditions that activate theHPI axis (i.e. crowding, shifts in pH, built up of ammonia andnitrates). Two parameters that could be analyzed in these paradigms aremortality and change in weight. Such parameters will demonstrate whetherthe ACTH analog of the invention is effective in lowering corticosteronelevels in fish.

1. A composition comprising an isolated adrenocorticotropic hormone(ACTH) analog peptide, the ACTH analog peptide comprising the peptide ofSEQ ID NO:20.
 2. The composition of claim 1, wherein the-administrationof the ACTH analog peptide in an in vivo Serum Corticosteroid InhibitionAssay reduces ACTH-induced corticosteroid secretion by at least 10%. 3.The composition of claim 1, wherein the ACTH analog peptide binds toadrenal membrane and displaces a peptide of SEQ ID NO:2 from adrenalmembrane, where the peptide binding is measured by an in vitroSerum-free Adrenal Competitive Binding Assay.
 4. The composition ofclaim 3, wherein the ACTH analog peptide binds to a MC-2R adrenalmembrane with at least a 2-fold greater affinity than the peptide of SEQID NO:2.
 5. The composition of claim 1, wherein the ACTH analog peptidereduces ACTH induced production of corticosterone by adrenal membrane inan in vitro Serum-free Adrenal Inhibition Assay.
 6. A compositioncomprising an isolated ACTH analog peptide, the ACTH analog peptidecomprising the peptide of SEQ ID NO:20, wherein the ACTH analog peptidebinds to adrenal membrane and displaces a peptide of SEQ ID NO:2 fromadrenal membrane, where the peptide binding is measured by an in vitroSerum-free Adrenal Competitive Binding Assay.
 7. The composition ofclaim 6, wherein the ACTH analog peptide reduces ACTH induced productionof corticosterone by adrenal membrane in an in vitro Serum-free AdrenalInhibition Assay.
 8. The composition of claim 6, wherein administrationof the ACTH analog peptide reduces ACTH-induced corticosteroid inductionby at least 10%, wherein corticosterone induction is measured by an invivo Serum Corticosteroid Inhibition Assay.
 9. The composition of claim6, wherein the ACTH analog peptide reduces ACTH induced production ofcorticosterone by adrenal membrane in an in vitro Serum CorticosteroidInduction Assay by at least 10% compared to the peptide of SEQ ID NO:2.10. The composition of claim 1, wherein the ACTH analog peptide consistsof the peptide of SEQ ID NO:20.
 11. A method of treating an ACTH-relatedcondition comprising the step of administering to a subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a peptide of SEQ ID NO:20, wherein thetherapeutically effective amount is effective to lower thecorticosteroid measured in the blood of the subject after theadministration of the pharmaceutical composition, and wherein theACTH-related condition is selected from the group consisting ofCushing's Syndrome and premature labor in which maternal plasmacorticotropin-releasing hormone levels are elevated.
 12. The method ofclaim 11, wherein the administration of the peptide of SEQ ID NO:20before or simultaneously with administration of ancorticosteroid-producing amount of an ACTH peptide comprising SEQ IDNO:2 reduces the serum corticosterone level measured after theadministration of the ACTH peptide compared to the serum corticosteronelevel measured without the administration of the peptide of SEQ IDNO:20.
 13. The method of claim 12, wherein the ACTH peptide is hACTH.14. The method of claim 11, wherein the administration of the peptide ofSEQ ID NO:20in an in vivo Serum Corticosteroid Inhibition Assay reducesACTH-induced corticosteroid secretion by at least 90%.
 15. The method ofclaim 11, wherein the ACTH-related condition is Cushing's Syndrome.